In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 μg, 40 μg, 8 μg, 1.6 μg, or 320 ng rat⁻¹ day⁻¹ for 12 days. The control group received corn oil only. DES effects were similar in the 8-μg group and higher dose groups and included significant (P ≤ 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-μg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the ≥8-μg groups and to a very low level in the 1.6-μg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 μg rat⁻¹ day⁻¹ for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-μg and 8-μg groups and 1 in the 1.6-μg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13–15 pups/litter in the controls. DES at a rate of 8 μg rat⁻¹ day⁻¹ for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-μg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.