To determine the optimal administration period for evaluation of ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD), VCD was intraperitoneally administered to female Sprague-Dawley rats at 0 (Control), 5, 20 and 80 mg/kg once a day for 2 or 4 weeks (2- or 4-week study). To identify small follicles, serial sections of the ovaries were stained with routine hematoxylin and eosin (HE) and proliferating cell nuclear antigen (PCNA) immunohistochemistry. In the 4-week study, decrease in small follicles was observed in the ovaries at 20 and 80 mg/kg. In the 2-week study, the same change was also observed at 80 mg/kg. Identification of small follicles using PCNA-stained slides was easier than that using HE-stained slides. In conclusion, histopathological findings in the ovaries are important for evaluation of female reproductive toxicity of VCD, and ovarian toxicity of VCD can be detected by administration for 2 weeks at an appropriate dose level. Furthermore, PCNA immunohistochemistry is effective for evaluation of small follicle destruction in chemical-induced ovarian toxicity.