Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N-acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha-lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta-N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.