The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le^x; Galβ4[Fucα3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888–1895). This glycan is recognized by ∼70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le^a; Galβ3[Fucα4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770–777). Herein, we test the hypothesis that Le^x- and Le^a-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le^x-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le^a-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le^x-containing neoglycoproteins. In contrast, neoglycoproteins containing β4-lactosamine (Galβ4GlcNAc), Lewis B (Fucα2Galβ3[Fucα4]GlcNAc), and sialyl-Le^x glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le^x- and Le^a-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le^x- and Le^a-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G_i protein. Thus, Le^x-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le^a-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.