The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females. A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (microm), tail DNA (%) and Olive tail moment (arbitrary units). To assess the effect of the estrogen 17beta-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells. The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.