The potential functions of testosterone and 5 alpha-dihydrotestosterone, the androgens normally present in human seminal plasma, on human spermatozoal physiology were evaluated by studying the effects of these two steroid hormones on the in vitro fertilizing capacity of human spermatozoa. Spermatozoa collected from presumably fertile men were washed in BWW medium and incubated with different concentrations (0, 100, 250, 500, 1000 pg/ml) of testosterone or 5 alpha-dihydrotestosterone for 5 hr before insemination of the zona-free hamster ova. Penetration of the zona-free hamster ova was scored 6 hr later and the results were analyzed statistically. Both testosterone and 5 alpha-dihydrotestosterone, at the concentrations tested, significantly decreased the in vitro penetration of the denuded hamster ova in comparison to the controls (p less than 0.05). A dose-dependent response was also observed for the 5 alpha-dihydrotestosterone tested. These findings indicate that exogenous testosterone and 5 alpha-dihydrotestosterone can inhibit the fertilizing capacity of human spermatozoa in vitro, and suggest that the androgens normally present in human seminal plasma may serve, in part, to prevent premature spermatozoal capacitation before the spermatozoa reach the site of fertilization in vivo.