The purpose of our research was to determine whether heparin-binding characteristics of human spermatozoa are related to fertilizing potential, as determined by the hamster egg in vitro penetration assay. Penetration rates were standardized (hamster egg in vitro penetration assay index) by comparison with semen from fertile controls in each bioassay. Saturation of heparin-binding domains was achieved in 100% of raw ejaculates (prewash), but in only 53% of "swim-up" (postwash) samples. The dissociation constants ranged from 0.31 to 48.75 nmol/10(6) cells, and binding site concentrations from 0.47 to 20.82 x 10(17) binding sites/cell. Heparin-binding affinity was significantly greater in prewash compared with postwash samples (p less than 0.01). In prewash samples the number of binding sites differed significantly between subjects having low and high penetration indices (5.67 +/- 1.05 vs 2.01 +/- 0.34 x 10(17) binding sites/cell, p less than 0.05). In prewash samples, binding affinity for heparin significantly correlated with hamster egg in vitro penetration assay indices (R2 = 0.142, p less than 0.05). In contrast, the number of binding sites in prewash samples was negatively correlated with hamster egg in vitro penetration assay indeces (R2 = 0.201, p less than 0.05). These data indicate that the heparin-binding assay may prove to be a rapid, sensitive, and inexpensive means of assessing fertilizing potential of human spermatozoa.