We have purified a sperm membrane protein, designated zonadhesin, that binds in a species-specific manner to the extracellular matrix (zona pellucida) of the egg, and cloned its cDNA. The cDNA encodes a novel protein with a single transmembrane segment separating a 36 amino acid, highly basic intracellular C terminus from a 2418-amino acid extracellular region. The extracellular sequence specifies a mosaic protein comprising a unique N-terminal domain, a mucin-like domain, and five tandem domains proximal to the membrane that are homologous to prepro von Willebrand factor. The N-terminal and mucin-like domains were absent from zonadhesin that bound to the egg extracellular matrix, suggesting that processing occurs during sperm maturation and/or capacitation. By Northern blotting and in situ hybridization, zonadhesin mRNA was detected only within the testis, where it was expressed primarily in haploid spermatids. The unique domain structure of zonadhesin suggests multiple functions, one of which is to mediate sperm adhesion to the zona pellucida.