To clarify genotoxic effects of occupational exposure to antineoplastic drugs in Japan, we examined DNA damage, assessed by the comet assay, in 121 female nurses and 46 female clerks working at three hospitals in the northeast of Japan. The comet assay is considered to be a sensitive and rapid method for DNA strand break detection in individual cells, and tail length and tail moment are used as the comet parameters. Concerning the basal characteristics, the 46 control subjects had higher rates of smoking and coffee-drinking habits and lower hemoglobin than the 121 nurses (p<0.05). The log-transformed tail length in the nurses was significantly longer than that in the control subjects after adjusting for possible covariates such as age and smoking habit (p<0.05). Also, the log-transformed tail length was significantly longer, in the 57 nurses who had handled antineoplastic drugs in the last six months, than that in the 46 control subjects (p<0.05); but, no significant difference in tail length or tail moment was seen between the two nurse groups with and without experience of handling hazardous drugs (p>0.05). These results suggest that Japanese nurses who have worked at hospitals using antineoplastic drugs may have a potential risk of DNA damage. To minimize this risk in Japan, use of biological safety cabinet and appropriate protective equipment, in addition to staff education and training, should be implemented in the healthcare environment.