OBJECTIVE Ketamine hydrochloride (KT) is a secondary amine that has been safely used as an injectable anesthetic and analgesic to avoid the production of nitroso compounds in the stomach. However, ketamine in the tablet form has recently become an abused, recreational drug. The aim of this study was to investigate the genotoxic effects of N-nitrosoketamine (NKT) and KT on the basis of an in vitro micronucleus (MN) test using a Chinese hamster lung fibroblast cell line (CHL/IU). METHODS NKT was synthesized from KT in our laboratory. In the MN tests, CHL/IU cells were continuously treated with either NKT or KT for 24, 48, or 72 hours without the S9 mix. The cells were also treated with NKT or KT with or without the S9 mix for 6 hours, followed by a recovery period of 18, 42, or 66 hours (short-term treatment). The results were considered to be statistically significant when the p-values of both Fisher's exact test and the trend test were less than 0.05. RESULTS After the short-term treatment with either NKT or KT with and without the S9 mix, the frequency of micronuclei significantly increased. However, the frequency of micronuclei did not significantly increase after the continuous treatment with either NKT or KT. Both NKT and KT were determined to be genotoxic in the short-term treatment with or without the S9 mix, but they were determined to be nongenotoxic in continuous treatment. CONCLUSIONS Our findings suggest that NKT has a stronger genotoxic effect than KT.