Previous studies have shown that RU486 decreases the concentration of intracellular calcium ([Ca2+]i) in human spermatozoa in vitro and partially antagonizes the effect of progesterone on calcium influx and sperm acrosome reaction. The present study has examined the effect of RU486 on the penetration of human spermatozoa into zona-free hamster oocytes. RU486 (10 microM) decreased significantly the rate of penetration of zona-free hamster oocytes by human spermatozoa, and nearly abolished penetration when present in the medium at concentrations of 50 and 100 microM. RU486 must be present in the medium to exert its inhibitory effect. At the same concentration (10 microM) as RU486, progesterone was unable to reverse the inhibitory effect of RU486 on the penetration rate of human spermatozoa.