The effect of human follicular fluid on human zona pellucida binding of spermatozoa was investigated using the hemizona binding assay (HZA). This effect was compared to that of progesterone, a known component of human follicular fluid. Exposure of spermatozoa to 25% pooled human follicular fluid for 1 h significantly reduced the number of spermatozoa bound to zona pellucida when compared to those without human follicular fluid treatment (149.1 +/- 30.7 versus 177.1 +/- 33.8, P < 0.01). The same phenomenon was observed after 3 h of treatment. The corresponding numbers of bound spermatozoa were 140.4 +/- 19.1 and 200.2 +/- 23.4 (P < 0.0001). Progesterone (1.0 microgram/ml) stimulated the zona pellucida-binding capacity of spermatozoa significantly under the same conditions (P < 0.01). The numbers of bound spermatozoa after 1 and 3 h progesterone treatment were 235.5 +/- 44.7 (control, 168.1 +/- 32.9) and 204.3 +/- 27.4 (control, 162.3 +/- 20.1) respectively. HZA comparing the effects of human follicular fluid and progesterone at concentrations equivalent to those found in human follicular fluid using matching hemizonae confirmed the inhibitory effect of human follicular fluid on sperm binding to zona pellucida (80.4 +/- 28.4 versus 149.8 +/- 35.2, P < 0.05). This inhibitory effect was also found in another eight individual human follicular fluid samples. Both human follicular fluid and progesterone did not affect the motility and viability of the treated spermatozoa when compared to the controls with the same incubation period. Although more spermatozoa underwent the acrosome reaction after 1 and 3 h of human follicular fluid treatment than in the control, the extent was comparable to those after progesterone treatment. These results suggested that human follicular fluid inhibited the zona pellucida-binding capacity of spermatozoa in vitro. This inhibitory effect of human follicular fluid was not mediated by progesterone, and did not result from the effects of human follicular fluid on sperm motility, viability and acrosome reaction.