The modulatory role of follicular fluid (FF) upon gonadotropin-induced granulosa cell (GC) differentiation has been previously demonstrated. In the present study, the stimulatory factor of large-antral FF (LFF) was partially characterized. Addition of ovine FSH to porcine GC culture increased 125I-hCG specific binding about 6-fold and progesterone (P4) secretion 15-fold. The FSH-induced 125I-hCG specific binding was increased 1.9-fold by LFF and decreased 7.7-fold in the presence of small-antral FF (SFF). It is suggested that SFF may contain an inhibitory factor which is overcome by a stimulatory factor accumulated in FF with follicular growth. An initial purification of LFF stimulatory factor was achieved by two-step ethanol elution. The obtained LFF purified fraction increased the FSH-induced 125I-hCG specific binding 1.5-fold and P4 secretion 4.3-fold compared to GC cultured with FSH + LFF. Addition of LH alone increased the P4 secretion 2.3-, 3.4-, and 6 fold in GC precultured with FSH alone, FSH + neat LFF, and FSH + LFF purified fraction, respectively, as compared to GC precultured in plain medium. Therefore, the LFF stimulatory increase of 125I-hCG specific binding may represent an increase of functional LH receptors. Freezing and thawing of LFF 4 times, exposing LFF to urea for 24 h, heating at 60 C for 20 min, and tryptic digestion each abolished the LFF stimulatory effect upon the FSH-dependent increase of 125I-hCG specific binding and P4 secretion. Thus, the LFF stimulatory activity could be attributed to one or more proteins.