This review has examined a number of issues surrounding the use of carboxylesterase activity in environmental monitoring. It is clear that carboxylesterases are important enzymes that deserve increased study. This class of enzymes appears to have promise for employment in environmental monitoring with a number of organisms and testing scenarios, and it is appropriate for inclusion in standard monitoring assays. Given the ease of most activity assays, it is logical to report carboxylesterase activity levels as well as other esterases (e.g., acetylcholinesterase). Although it is still unclear as to whether acetylcholinesterase or carboxylesterase is the most "appropriate" biomarker, there are sufficient data to suggest that at the very least further studies should be performed with carboxylesterases. Most likely, data will show that it is optimal to measure activity for both enzymes whenever possible. Acetylcholinesterase has the distinct advantage of a clear biological function, whereas the endogenous role of carboxylesterases is still unclear. However, a combination of activity measurements for the two enzyme systems will provide a much more detailed picture of organism health and insecticide exposure. The main outstanding issues are the choice of substrate for activity assays and which tissues/organisms are most appropriate for monitoring studies. Substrate choice is very important, because carboxylesterase activity consists of multiple isozymes that most likely fluctuate on an organism- and tissue-specific basis. It is therefore difficult to compare work in one organism with a specific substrate with work performed in a different organism with a different substrate. An attempt should therefore be made to standardize the method. The most logical choice is PNPA (p-nitrophenyl acetate), as this substrate is commercially available, requires inexpensive optics for assay measurements, and has been used extensively in the literature. However, none of these beneficial properties indicates that the substrate is an appropriate surrogate for a specific compound, e.g., pyrethroid-hydrolyzing activity. It will most likely be necessary to have more specific surrogate substrates for use in assays that require information on the ability to detoxify/hydrolyze specific environmental contaminants. The use of carboxylesterase activity in TIE protocols appears to have excellent promise, but there are further technical issues that should be addressed to increase the utility of the method. The main concerns include the large amount of nonspecific protein added to the testing system, which can lead to undesirable side effects including nonspecific reductions in observed toxicity, decrease in dissolved oxygen content, and organism growth. It is probable that these issues can be resolved with further assay development. The ideal solution would be to have a commercial recombinant carboxylesterase that possessed elevated pyrethroid-hydrolysis activity and which was readily available, homogeneous, and inexpensive. The availability of such an enzyme would address nearly all the current method shortcomings. Such a preparation would be extremely useful for the aquatic toxicology community. Further work should focus on screening available esterases for stability, cost, and activity on pyrethroids, with specific focus on esterases capable of distinguishing type I from type II pyrethroids. It would also be beneficial to identify esterases that are not sensitive to OP insecticides. Many esterases and lipases are available as sets to test chemical reactions for green chemistry, enabling large-scale screening. Other potential approaches to increase the utility of the enzyme include derivatization with polyethylene glycol (PEG) or cyanuric acid chloride to increase stability and reduce microbial degradation. It is also possible that the enzyme could be formulated in a sol gel preparation to increase stability. It is likely that the use of carboxylesterase addition will increase for applications in sediment TIEs. Carboxylesterases are an interesting and useful enzyme family that deserves further study for applications in environmental monitoring as well as to increase our understanding of the fundamental biological role(s) of these enzymes. There are, of course, other enzymes that show high esterase activity on pyrethroids but are not technically carboxylesterases in the alpha/beta-hydrolase fold protein family. These enzymes should also be examined for use in TIE protocols and "esterase" arrays as well as for general applications in environmental monitoring. One can envision the creation of a standardized screen of enzymes with esterase activity to (1) identify environmental contaminants, (2) estimate the potential toxic effects of new compounds on a range of organisms, and (3) monitor organism exposure to agrochemicals (and potentially other contaminants). This approach would provide a multibiomarker integrative assessment of esterase-inhibiting potential of a compound or mixture. In conclusion, much is still unknown about this enzyme family, indicating that this area is still wide open to researchers interested in the applications of carboxylesterase activity as well as basic biological questions into the nature of enzyme activity and the endogenous role of the enzyme.