The effects of ethanol on the loss of the human sperm acrosome, as determined by the chlortetracycline fluorescence assay and by indirect immunofluorescence assay, were assessed over 6 hours during incubation at 37 C in BWW medium containing 0 to 250 mM ethanol. Both assays gave the same results. At the end of 6 hours, 48 +/- 6% acrosomal loss was found in samples in 250 mM ethanol compared with 4 +/- 1% in the absence of ethanol. After 0.25 hour, the first time point chosen for sampling, the spermatozoa in 250 mM ethanol showed 23 +/- 3% loss of acrosomes compared with less than 1% in the absence of ethanol. Ultrastructural studies revealed that the ethanol-treated spermatozoa showed complete acrosomal loss as well as loss of the equatorial segment. No examples of the vesiculation characteristic of the physiologic acrosome reaction were found in the 150 cells examined. Calcium is required for the ethanol-mediated acrosomal loss: omission of Ca2+, addition of 2 mM EGTA, or 0.2 mM verapamil blocked the effect. Ethanol induced a dose-dependent efflux of cholesterol from human spermatozoa, but the ethanol-induced acceleration of acrosomal loss occurred to the same extent in the presence of cholesterol microdispersions that prevented this efflux. The loss of the equatorial segment, which is necessary for egg penetration, during ethanol-induced acrosomal loss would explain the known effect of ethanol in inhibiting, rather than enhancing, the penetration of zona-free hamster eggs by human spermatozoa.