An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role of O-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminal N-acetylglucosaminyl or, alternatively, a terminal alpha-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit the in vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAc beta 1,4GlcNAc beta 1,4GlcNAc,was inactive (1-72 microM range). In contrast, a beta 4-galactosyl-capped trisaccharide (Gal beta 1,4GlcNAc beta 1, 4GlcNAc) and an alpha 3-galactosyl-capped trisaccharide (Gal alpha 1,3Gal beta 1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 microM and 5.3 microM, respectively). The addition of an alpha 3-fucosyl residue to each of these two competitive inhibitors, forming Gal beta 1,4[Fuc alpha 1,3] GlcNAc beta 1,4GlcNAc or Gal alpha 1,3Gal beta 1, 4[Fuc alpha 1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nM). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the alpha 3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated beta-galactosyl-capped trisaccharide.