A glycolipid linked high-affinity folate binding protein (FBP) is present in human semen at a concentration of 1-2 nmol/L. The association between FBP and seminal components as well as the cellular source of FBP was analysed. Immunoblotting of human seminal plasma, with and without prostasomes, prostasomal fractions and spermatozoa with antibodies against human FBP revealed a single distinct band similar to that observed with purified human FBP. Flow cytometry identified FBP on the surface of ejaculated spermatozoa. Immunohistochemistry showed positive immunostaining of epididymal epithelium, vas deferens and ejaculated spermatozoa, whereas the prostate gland, seminal vesicles, testicular spermatozoa and seminiferous tubules of testis stained negatively. Electron microscopy immunocytochemistry with antibodies against rat FBP showed labelling located to luminal microvilli and intracellular vesicles of rat epididymal epithelial cells and the surface of spermatozoa in the epididymal duct. High-affinity binding of picomolar amounts of tritiated folate to fractions of human prostasomes or prostasome-like vesicles was completely depressed by excess amounts of unlabelled folate. The study indicates that FBP is secreted from the epithelia of epididymis and vas deferens, and that a small fraction of FBP is associated with prostasome-like vesicles which adhere to spermatozoa in the epididymal duct. FBP could have a bacteriostatic function by depriving folate-requiring bacteria of folate and/or ascertain a normal DNA replication subsequent to fertilization by vectorial transfer of folate to the inner compartment of the spermatozoa.