Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le^x)-containing glycan (Galβ4[Fucα3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888–1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le^x-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa₅₆₈)-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC₅₀ ∼180 nM), which at saturation, reduced Alexa₅₆₈-ZP3 binding by ∼70%. A Lewis A (Le^a)-capped glycan (Galβ3[Fucα4]GlcNAc) was also a potent inhibitor (IC₅₀ ∼150–200 nM), but at saturation, it reduced Alexa₅₆₈-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAcβ4 or Galβ4 residues did not compete; neither did sialyl-Le^x (Neu5Acα 3Galβ4[Fucα3]GlcNAc-Lewis X) nor sulfo-Le^x (3′-O-SO₃-Lewis X). However, at saturation, Galα3Galβ4GlcNAcβ3Galβ4Glc reduced Alexa₅₆₈-ZP3 binding by ∼70% but with moderate apparent affinity (IC₅₀ ∼3000 nM). Fluorescence microscopy revealed that Alexa₅₆₈-labeled Le^x-Lac-BSA, Le^a-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le^a-Lac did not inhibit binding of Alexa₅₆₈-Le^x-Lac-BSA, and Le^x-Lac did not inhibit binding of Alexa₅₆₈-Le^a-Lac-BSA. Finally, Le^x-Lac and Le^a-Lac had an additive inhibitory effect on Alexa₅₆₈-ZP3 binding. Thus, Le^x is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le^a binding defines a different but less-abundant class of sites.