Inhibin activity from human follicular fluid was purified by successive chromatographies on Sephadex G-50, DEAE-Biogel A and orange A dye matrix. Inhibin activity was associated only with the protein(s) that bound to orange A (OrA-2). Daily injection of OrA-2, 1 h prior to hMG into 10-day-old female rats for 4 days caused a significant inhibition of hMG-induced estradiol secretion. In vitro, OrA-2 dose-dependently inhibited the amounts of estradiol secreted by porcine granulosa cells during a 3-h incubation. Orange A-unbound proteins, on the other hand, induced a dose-dependent increase in estradiol as well as progesterone secretion by porcine granulosa cells in vitro. Separation of stimulator from the inhibitor by orange A chromatography led to an increase in the relative inhibin activity (25-50-fold) as well as aromatisation-suppressing activity (60-fold). The results indicate a possible local action of hFF inhibin to regulate aromatisation activity.