Pesticides often cause environmental pollution and adverse effects on human health. We have chosen four structurally different pesticides (endosulfan, an organochlorine pesticide; chlorpyrifos, an organophosphate insecticide; cypermethrin, type II pyrethroid insecticide, and 2,4-dichlorophenoxyacetic acid, a chlorinated aromatic hydrocarbon acid pesticide) to examine and compare their effects on DNA damage in acutely cultured human lymphocytes by the comet assay. In addition, possible differences in response between smoking and nonsmoking subjects were also investigated. Venous blood samples were obtained from healthy male nonsmoker (n = 7) and smoker (n = 8) donors. Primary cultures of lymphocytes were prepared and test groups were treated with three different concentrations (1, 5, and 10 mu M) of endosulfan, chlorpyrifos, cypermehrin, and 2,4-D. DNA damage was assessed by alkaline comet assay. We determined an increase in the ratio of DNA migration in human lymphocyte cell cultures as a result of treatment with cypermethrin, 2,4-D and chlorpyrifos at high concentration. Endosulfan had no significant genotoxic effect even at 10 mu M concentration. We suggest that chlorpyrifos and cypermethrin are more potentially genotoxic than endosulfan and 2,4-D. Our findings also indicate that the only significant DNA damage between smokers and nonsmokers was observed in the 2,4-D-treated group. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol 26: 433-442, 2011.