To evaluate the role of nonsteroidal, follicular fluid proteins in folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled porcine follicular fluid (PFF) was dialyzed against 0.025 M Tris/HCl, pH 7.5, using 10,000 molecular weight exclusion membranes, then passed through agarose-immobilized textile dye. Activity was determined by test fraction inhibition of human menopausal gonadotropin (hMG) [2 U human luteinizing hormone (LH)/follicle-stimulating hormone (FSH) per day] induced ovarian weight and serum estradiol increase in hypophysectomized, diethylstilbestrol (DES)-treated, 25-day-old female rats. Specific inhibition (84 +/- 7.4%) of ovarian weight increase was found in the material (5 ml) eluted from the orange A column with KCl (1.5 M, pH 6.8). Inhibitory activity of the orange A-bound material which eluted through a standardized Sephadex G-100 column corresponded to a molecular weight of 12,000-30,000. Isoelectric focusing (IEF) on a Sephadex G-15 support bed of orange A-bound material demonstrated inhibitory activity at pH 3.7-4.0. Serial dilutions of active material from IEF preparations demonstrated a dose-response relationship in the bioassay. No demonstrable activity was found in similar fractions eluted through a Concanavalin A-Sepharose 4B column with or without addition of alpha-methyl mannoside (2 M, pH 7). When active fractions were heated (56 degrees C, 1 h) or exposed to trypsin (10 mg%), activity was lost. When aliquots of the saturated ammonium sulfated precipitated, dialyzed, orange A-bound, Sephadex G-100 (Ve/Vo 1.3-1.7) eluent were separated by high-performance liquid chromatography (HPLC) using gel exclusion columns, activity in the bioassay was recovered in the 18,000-35,000 molecular weight range.(ABSTRACT TRUNCATED AT 250 WORDS)