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The present studies examined the inhibitory effect of mid-cycle and preovulatory bovine follicular fluid (bFF) on the secretion of progesterone and prostaglandin F₂ (PGF₂) by bovine theca and granulosa cells cultured in tissue culture Medium 199. The inhibitory effect of bFF on the secretion of PGF₂ by incubated and cultured hemipituitary glands of male rats was also studied. Addition of 5, 10 or 20% charcoal-extracted, mid-cycle bFF to the culture medium caused a twofold decrease in the accumulation of both progesterone and PGF₂ by the cultured granulosa cells (P < 0·01). In contrast, when preovulatory bFF was used, there was no inhibitory effect on secretion of progesterone and PGF₂. The addition of 10% mid-cycle bFF to the culture medium caused a two- to fourfold decrease in the accumulation of both PGF₂ and progesterone by the cultured theca (P < 0·008). However, in the presence of 1 µg LH/ml, the inhibitory effect of mid-cycle bFF on progesterone and PGF₂ secretion was abolished. The secretion of PGF₂ was significantly (P < 0·03) decreased in male rat hemipituitary glands after 5 h of incubation or 18 h of culture. These findings suggest that bFF from mid-cycle follicles inhibits prostaglandin synthetase as well as luteinization. The inhibition disappeared with bFF from preovulatory follicles.