In this study, genotoxic activities of four halogenated anesthetics (halothane, isoflurane, sevoflurane and desflurane) were investigated in human peripheral blood lymphocytes (PBLs) and sperm cells in vitro by alkaline comet assay. For this purpose, sperm or lymphocyte suspension was exposed to different concentrations (0.1 mM, 1 mM, 10 mM and 100 mM) of anesthetic agents and 1% dimethyl sulfoxide (DMSO) or phosphate-buffered saline (PBS) as controls. The DNA strand breaks as well as alkali-labile sites were measured as percentage tail intensity with comet assay. The results of this study demonstrate that all analyzed drugs were capable of inducing DNA damage on PBLs in a dose-dependent manner in vitro. However, the results in sperm cells were slightly different since we did not observe any genotoxic effect for desflurane in any of the exposure doses, and the genotoxic effect of halothane was not dose dependent. This experimental study points out to the presence of DNA damage after exposure to halogenated anesthetics in both PBLs and sperm cells, although this effect seems to be higher in PBLs.