A group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), are the major proteins found in bovine seminal fluid. These proteins are secretory products of seminal vesicles, and they bind to spermatozoa upon ejaculation, suggesting that there are binding sites for these proteins on the spermatozoa. It was of interest to characterize these binding sites on spermatozoa which may help in the elucidation of the biological function of BSP proteins. The binding sites on spermatozoa are resistant to protease or acid treatment and are heat-stable but extractable with organic solvents. The solvent-extractable material, when coated on plastic microtitration wells, binds radiolabeled BSP proteins thus indicating the lipid nature of the BSP binding sites on spermatozoa. We investigated the specificity of interaction of BSP proteins with lipids using liposomes of phospholipids, solid-phase, and thin-layer chromatography-overlay techniques. Results showed that BSP-A1, -A2, and -A3 proteins bound specifically to those phospholipids which contain the phosphorylcholine group. In contrast, BSP-30-kDa protein preferentially bound to phospholipids containing the phosphorylcholine moiety but also interacted with phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Furthermore, of those lipids that were extracted from spermatozoa, only phospholipids which contain the phosphorylcholine moiety bound radiolabeled BSP proteins. These data suggest that the BSP protein binding sites on spermatozoa are phospholipids. We propose that this specific interaction plays an important role in the membrane modification of spermatozoa that occurs during capacitation and/or acrosome reaction.