In a previous study we reported the presence in human follicular fluid (hFF) of a FSH receptor-binding inhibitor (hFSH-BI) with FSH agonist activity, which was immunologically similar to FSH but could be distinguished from FSH on the basis of its greater stability in acid. We have now purified hFF-derived hFSH-BI after molecular sieving on Sephracyl S-100 ion exchange chromatography using Diethyl-aminoethyl-cellulose followed by polyacrylamide gel electrophoresis (PAGE). The purified hFSH-BI had a potency approximately 12,000-fold greater than that of dialyzed hFF, based on its ability to inhibit the binding of [125I]hFSH to its membrane receptor. The purified hFSH-BI also had FSH agonist activity, stimulating estradiol synthesis in cultured rat Sertoli cells. Upon sodium dodecyl sulfate (SDS)-PAGE, hFSH-BI migrated as two bands of almost identical mobility, with an estimated mol wt of 57,000, compared with 30,000 for pituitary FSH run simultaneously. A monoclonal antibody to hFSH that also recognizes hFSH-BI was used for Western blot analysis of the SDS-PAGE fraction. The Western blot confirmed the detection of two bands with very similar mobilities and estimated mol wt of 57,000, which were clearly distinguishable from that of immunologically reactive hFSH run in parallel. The hFSH-BI bands showed similar profiles upon cyanogen bromide cleavage and had indistinguishable amino acid compositions. The amino acid composition of hFSH-BI was clearly distinct from those of hFSH, hLH, hCG, and the alpha-subunit of human inhibin. Our studies confirm the presence in hFF of a unique agonist protein which is of potential importance in the regulation of gonadal function.