In the ovary, cortisol is oxidized to cortisone by 11β-hydroxysteroid dehydrogenase (11βHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11βHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP -dependent 11βHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% ± 2.3% and 72.8% ± 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP -dependent 11βHSD activities (111.5% ± 21.6% and 55.2% ± 5.7% respectively). Hydrophobic compounds inhibited NADP -dependent cortisol oxidation by 58.2% ± 5.1% (bovine) and 45.7% ± 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11βHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD -dependent cortisol oxidation. The ovarian modulators of NADP -dependent 11βHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11βHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E₂ and PGF_2α. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP -dependent 11βHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11βHSD stimulus accompanied by increased content of the 11βHSD inhibitors, exerts a net inhibitory effect on 11βHSD activity.