The effect of exogenous prolactin (PRL) on the fertilizing capacity of human spermatozoa in vitro was evaluated by the zona-free hamster ova penetration assay. Bovine and ovine PRL, in both physiological and pharmacological concentrations, failed to increase the penetration after 5 hr of incubation in vitro. The percent motility of spermatozoa was not influenced by PRL during the incubation period. Exogenous PRL does not affect the fertilizing capacity of human spermatozoa in vitro. The role of prolactin, normally present in seminal plasma and female reproductive tract fluid, in the human spermatozoal capacitation process merits further investigation.