The aim of this study, based on a prospective analysis, was to evaluate the accuracy of the TEST-yolk buffer enhanced hamster egg penetration test (HEPT) and acrosin activity in discriminating fertile from infertile males as compared with the conventional semen profile. The incidence of spontaneous pregnancies was monitored during a follow-up period ranging between 18-24 months in 62 couples without untreatable female infertility. The diagnostic accuracy of the HEPT was significantly higher than that of the conventional semen profile, as evaluated with fitted ROC curves. The best discriminatory ability was exhibited by a penetration index (penetrations/oocyte) of 2 as cut-off value. In our series it gave a positive predictive value of 77.8% and a negative predictive value of 92.3%. Acrosin activity did not show a discriminating power significantly higher than the conventional semen profile, but an acrosin activity <20 microIU/10(6) spermatozoa was found only among patients who did not achieve a pregnancy. In conclusion, this prospective analysis indicates that the TEST-yolk buffer enhanced HEPT constitutes a significant improvement over the conventional semen profile in discriminating fertile from infertile males. Acrosin activity could provide a useful adjunctive test because of the high negative predictive value of its poor results.