The rodent Hershberger assay proposed by the Organization for Economic Co-operation and Development (OECD) is in the process of the validating a test method to detecting the androgenic or anti-androgenic compounds. The aim of this study was to compare the anti-androgenic properties of flutamide, vinclozolin, procymidone, linuron, and p,p'-DDE in a 10-day Hershberger assay. In the present study, we used immature Sprague-Dawley male rats castrated at 6 weeks of age. Testosterone propionate (TP) was subcutaneously injected for 10 consecutive days at doses of 0.1, 0.2, 0.4, 0.8, or 1.6 mg/kg per day. To compare the anti-androgenic activity of test compounds, flutamide (1, 5, 10, or 20 mg/kg per day), a pure androgen antagonist was used as a positive control, and administered by oral gavage after TP (0.4 mg/kg per day) treatment. In addition, vinclozolin (25, 50, or 100 mg/kg per day), procymidone (25, 50, or 100 mg/kg per day), linuron (25, 50, or 100 mg/kg per day), and p,p '-DDE (25, 50, or 100 mg/kg per day) were also administered by oral gavage after TP (0.4 mg/kg per day) treatment. As expected, TP dose-dependently increased accessory sex organ weights, and statistically significant effects were observed at doses of 0.1 (only seminal vesicles) or 0.2mg/kg per day and above. Serum testosterone levels increased significantly at 0.4 mg/kg per day and above, while serum LH levels were decreased in a dose-dependent manner. Flutamide significantly inhibited the TP-induced re-growth of seminal vesicles, ventral prostate, and Levator ani plus bulbocavernosus muscles (LABC) at 1mg/kg per day and above, and Cowper's glands and glans penis at 5mg/kg per day and above. In contrast to accessory sex organ weights, flutamide did not affect the serum testosterone levels compared to the control at any concentration, but serum LH levels were significantly increased at doses of 10 and 20 mg/kg per day. Similar to flutamide, vinclozolin caused a statistically significant decrease in the weights of seminal vesicles (to 65 and 40% of the control), ventral prostate (to 66 and 51% of the control), LABC (to 81 and 66% of the control), and Cowper's glands (to 81 and 65% of the control) at 50 and 100 mg/kg per day, respectively. Glans penis weight was also significantly reduced (to 79% of the control), but only at 100 mg/kg per day. The most pronounced effects were observed in the procymidone treatment groups. Procymidone significantly inhibited TP-induced re-growth of accessory sex organs at 25mg/kg per day and above, whereas glans penis weight significantly decreased (to 69% of the control), but only at 100 mg/kg per day. Linuron also inhibited TP-induced re-growth of the seminal vesicles (to 72 and 53% of the control), ventral prostate (to 75 and 62% of the control), Cowper's glands (to 74 and 61% of the control) at 50 and 100 mg/kg per day, respectively. LABC (to 65% of the control) and glans penis (to 80% of the control) weights were significantly reduced, but only at 100 mg/kg per day. In case of p,p'-DDE, seminal vesicle weights were significantly decreased at 50 (to 66% of the control) and 100 mg/kg per day (to 58% of the control). In addition, ventral prostate (to 79% of the control), LABC (to 75% of the control), and Cowper's gland (to 82% of the control) weights were reduced, but only at 100 mg/kg per day. On the contrary, no statistically significant differences in serum testosterone or LH levels were observed versus the control. p,p'-DDE significantly increased liver weight in a dose-dependent manner, without affecting on body weights. Our results indicate that procymidone may act as a stronger androgen receptor (AR) antagonist than vinclozolin, linuron, or p,p'-DDE. We conclude that the 10-day Hershberger assay is a sensitive method for detecting potential anti-androgenic compounds.