Mammalian spermatozoa undergo the acrosome reaction (AR) in response to the interaction of a carbohydrate-recognizing molecule(s) on the sperm plasma membrane (sperm surface receptor) and its complementary glycan (ligand) moiety(ies) on the zona pellucida (ZP). Previously, we demonstrated that a hexose (mannose) or two amino sugars (glucosaminyl or galactosaminyl residues) when covalently conjugated to a protein backbone (neoglycoproteins) mimicked the mouse ZP3 glycoprotein and induced the AR in capacitated mouse spermatozoa (Loeser and Tulsiani, Biol Reprod 1999; 60:94-101). To elucidate the mechanism underlying sperm-neoglycoprotein interaction and the induction of the AR, we have examined the effect of several AR blockers on neoglycoprotein-induced AR. Our data demonstrate that two known L-type Ca(2+) channel blockers prevented the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA). The fact that the L-type Ca(2+) channel blockers (verapamil, diltiazem) had no inhibitory effect on sperm surface galactosyltransferase or alpha-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.