We studied the effects of active factors present in bovine follicular fluid (bFF) from large healthy or atretic follicles on steroidogenic capability of cultured bovine granulosa cells. Pools of bFF were collected from follicles (> 10 mm; abattoir material) and classified individually as being healthy (bFF-healthy) or atretic (bFF-atretic). Pools of jugular plasma were used as controls and were from heifers bled during the growing (plasma-growing) or the regressing (plasma-regressing) phase of follicular dominance. Granulosa cells were cultured in serum-free conditions and under minimal FSH support (.5 ng/mL) for the first 3 d in order to maintain their physiological estradiol production in response to FSH. Effects of addition of bFF and plasma at final concentrations of 0, 1, or 5% on estradiol and progesterone production and on the percentage of apoptotic cells were determined on d 4 of culture following stimulation of granulosa cells with either 2 or 6 ng/mL FSH. In a parallel experiment, evaluation of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity was measured following addition of bFF. In contrast to plasma, addition of bFF pools decreased (P < .001) the FSH-induced estradiol production. Such suppression occurred in a dose-related manner (P < .05) and to a greater extent (P < .001) following addition of bFF from atretic than from healthy follicles. The FSH-induced progesterone production was not affected (P > . 1) by addition of bFF but was stimulated (P < .05) by that of plasma. Follicle-stimulating hormone decreased (P < .001) the percentage of apoptotic granulosa cells and this effect was further enhanced (P < .001) by addition of 1 or 5% bFF. The source of bFF did not affect (P > . 1) the percentage of apoptotic cells measured at the end of the culture period. On d 4, treatment with bFF increased (P < .001) granulosa cell androstenedione conversion into testosterone. Results of the present study indicate that factors contained in bFF can suppress granulosa cell estradiol production, and the suppressive effect varies according to the degree of atresia of the follicle from which the fluid has been harvested.