Studies on topical repellent efficacy conducted with caged mosquitoes in the laboratory are important to both the development and regulation of insect repellents. Guidelines for laboratory studies stipulate specific densities, sex ratios, and biting rates, whereas those for field studies are governmentally required before a promising repellent can be registered for human use. These protocols stipulate minimum biting rates alone. Relatively little is known, however, about the influence of mosquito density and sex ratio on their biting propensity, either in the field or laboratory. Using Environmental Protection Agency guidelines for cage testing, we studied the influence of mosquito density and sex ratio in laboratory repellency tests of the biopesticide Ethyl butylacetylaminopropionate (IR3535™) (20%) against three mosquito species (Aedes aegypti, Anopheles aquasalis, and Culex quinquefasciatus). DEET [3-(N-acetyl-N-butyl) aminopropionic acid ethyl ester] (20%) in a laboratory prepared formulation was used as a comparison article. Studies were conducted by trained investigators at the BioAgri Laboratories in Brazil. We found that higher mosquito density generally decreased protection time, but that the influence of sex ratio was more complex. The presence of male mosquitoes increased protection times against Aedes and Anopheles perhaps because mate-seeking males interfered with female feeding. Interestingly, by contrast, protection times decreased against Culex in the presence of males. Such considerations may potentially assist in improving the match between cage and field testing under a broader range of conditions that permit more accurate labeling of repellents for safe and effective use by consumers.