The comet test (Single Cell Gel Electrophoresis or SCGE) is an easy to perform, rapid and highly sensitive genotoxicity assay; it requires small amounts of biological substrate and is applicable in vivo and in vitro to a wide variety of cells and tissues. Modified versions of the comet test able to detect oxidative damage or the effects of agents inducing DNA-DNA or DNA-protein crosslinks are available. Similarly to other genotoxicity assays, the comet test is not predictive far individual cancer risk. Limitations and critical features presently linked to comet test applications, with particular regard to the biomonitoring of individuals exposed to genotoxic agents, include: lack of sensitivity with respect to aneugens (agents inducing numerical chromosomal aberrations), possible underestimation of genotoxic potency of agents with mixed action mechanisms, sensitivity depending on the genotoxic agent itself, dependence an biological substrate with regard to the influence of cytotoxicity on the assay results, influence of age, tobacco smoke, alcohol and drug consumption, diet, kinetics of DNA adducts and DNA repair mechanisms. Consequently, a routine use of the comet test in biological monitoring of individuals that are occupationally and environmentally exposed to genotoxic agents is submitted to its validation by multicentric studies on large population samples with different exposure patterns, with a suitable characterisation of the role played by the above mentioned factors. Human studies should also be increasingly focused on the direct cellular targets of exposure to genotoxicants (e.g. epithelial cells of oral cavity and airways).