Fertilin alpha and beta (previously known as PH-30 alpha and beta) are sperm surface proteins implicated in sperm-egg binding and membrane fusion. In this work we report the purification, characterization, and cloning of the bovine homologues of fertilin alpha and beta. Immunofluorescence and Western blotting, using polyclonal antibodies made against guinea pig fertilin, identified likely bovine fertilin alpha and beta homologues. These candidates were purified under nondenaturing conditions using lectin and ion-exchange chromatography and were subjected to amino acid sequence analysis. Degenerate oligonucleotides made from bovine N-terminal protein sequences and from guinea pig fertilin sequences were used to isolate clones encoding bovine fertilin alpha and beta from a bovine testis cDNA library. The predicted proteins have the same domain organization as guinea pig fertilin alpha and beta: pro, metalloprotease-like, disintegrin-like, and cysteine-rich domains, followed by an EGF-like repeat, a transmembrane domain, and a cytoplasmic tail. In contrast to the reported sequence of guinea pig fertilin alpha, the mature, proteolytically processed bovine fertilin alpha subunit contains a complete disintegrin-like domain, and a potential amphipathic beta strand, which may be involved in sperm-egg fusion. Gradient sedimentation experiments suggest that the fertilin alpha/beta heterodimer may be present on the sperm surface as a higher-order oligomer.