The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.