Porcine follicular fluid (PFF) inhibited the binding of 125I-human follicle-stimulating hormone (hFSH) to receptor in vitro in a dose-dependent fashion. PFF (2.5 l) was fractionated on the basis of apparent molecular weight (Mr) by ultrafiltration using hollow fibers and membranes of precalibrated pore size. Desalted, low Mr (500-5000) subfractions containing FSH-binding inhibitor (FSH-BI) activity were further purified by Sephadex G10 gel filtration and anion-exchange high-performance liquid chromatography (HPLC). This resulted in the partial purification of several low Mr FSH-BIs. Three major peaks of FSH-BI were resolved on the Sephadex G10 column eluted with water; G10-1 [elution volume (Ve)/exclusion volume (Vo) = 1.1] had only FSH-BI activity, while G10-2 (Ve/Vo = 1.4) and G10-3 (Ve/Vo = 1.5) had both FSH-BI and luteinizing hormone (LH)-BI activities. A fourth strongly retarded peak (G10-4; Ve/Vo = 2.7) was also obtained. This latter fraction had only FSH-BI activity and represented less than 1% of the FSH-BI activity applied to the column. No separation of these fractions was obtained when the column was eluted with 10 mM ammonium acetate instead of water, suggesting resolution was due to ion-exchange or hydrophobic interactions with the Sephadex. Anion-exchange (Polyanion SI) HPLC of G10-1, G10-2 or G10-3 samples resolved several fractions with FSH-BI activity. A fraction unretained at either pH 5.0 or 7.0 (HPLC-1) was present in all samples. A fraction strongly retained by the column (HPLC-2) and a fraction eluted between 0.13 to 0.24 M acetate (HPLC-3) were present in G10-1 and G10-2 but not in G10-3. HPLC-4, eluted between 0.32 to 0.36 M acetate at pH 5.0, was detected only in G10-3 samples. The most potent low Mr FSH-BI obtained (HPLC-2) inhibited FSH binding by 50% at a dose of 10 micrograms and was enriched approximately 2500-fold relative to whole follicular fluid. These results indicate that PFF contains several low (500-5000) Mr inhibitors of FSH binding to receptor in vitro which differ on the basis of charge, hormone specificity and possibly molecular size and hydrophobicity.