SVS VII, one of seven major proteins in mouse seminal vesicle secretion, was purified to homogeneity. Neither glycoconjugate nor free thiol group was detected in the protein. The primary structure deduced from the corresponding cDNA was confirmed using amino acid sequence determination, which supported the finding that SVS VII consists of 76 amino acid residues with five disulfide bridges. Accordingly, it has a theoretical molecular mass of 8538, which was proven using the mass spectrum of SVS VII. The CD spectrum of SVS VII in 50 mm phosphate buffer at pH 7.4 appeared as one negative band arising from the beta form at 217 nm and several fine structures due to nonpeptide chromophores including a prominent band for the disulfide bond at 250 nm. This, together with the predicted secondary structures, indicated no helices but a mixture of beta form, beta turn, and unordered form in SVS VII. A cytochemical study illustrated the presence of the SVS VII-binding region on the entire surface of mouse sperm. The SVS VII-sperm binding was inhibited by the dispersed sperm lipids. The results of TLC overlay assay for the binding of (125)I-SVS VII to phospholipids and the interaction between SVS VII and phospholipid liposomes demonstrated a specific binding of this protein to both phosphatidylethanolamine and phosphatidylserine. The SVS VII-sperm binding greatly enhanced sperm motility but did not induce sperm capacitation. Heating the protein solution for 10 min at 90 degrees C unfolded the protein molecule, and the unfolded SVS VII immobilized the sperm.