To confirm the applicability of MTT assay and sperm quality analyzer (SQA) to the examination of sperm viability and motility in rats, epididymal spermatozoa derived from male rats (Slc:SD) treated with nitrobenzene at a dose of 60 mg/kg/day for 16 days were examined by these methods. Epididymal fluid was suspended in Dulbecco's modified Eagle's medium containing 25 mM fructose in ratio of 1:125. Sperm concentrations in the epididymal fluid were 2.48 ± 0.55 (× 106/μl, mean ± SD, n = 8) in the control and 2.20 ± 0.54 in the nitrobenzene group, and did not significantly differ between the groups. Both absorbance unit of the MTT assay (1.497 ± 0.406 vs. 1.084 ± 0.350) and SMI (sperm motility index) value of the SQA (204.0 ± 15.3 vs. 182.5 ± 21.6) were significantly decreased in nitrobenzene group. The results suggest that the examinations of sperm viability and motility with MTT assay and SQA are available for the reproductive toxicology in rats.