Cytotoxic effects of nitrobenzene (NB) on spermatogenesis of mature Sprague-Dawley (Crj:CD) rats were analyzed by measuring the DNA content distribution and testicular weight at 1, 2, and 3 weeks of daily oral dose of NB (60 mg/kg/day). Rats at the age of 9 weeks were used because the ratios of 1C, 2C, S, 4C to total testicular cells were stabilized after the age of 52 days. Within a week of administration, a large number of 1C cells were lost but 2C cells proliferated, resulting in little change of testicular weight. In another week that followed, the number of 1C cells and testicular weight were decreased, but the ratio of S-4C cells was increased, indicating that an appreciable number of 2C cells could progress to the 4C compartment. The data indicated that (1) 1C cells were destroyed, and (2) meiosis of secondary spermatocytes was suppressed, but (3) NB had little effect on the spermatocytes prior to the early pachytene stage. This interpretation was reinforced by the observation that (4) the ratio of 1C cells returned to a nearly normal level during a recovery period of 2 weeks. In conclusion, flow cytometry could offer an efficient method for the quantitative analysis of male reproductive toxicity.