The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E₂), and progesterone (P₄) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10^-7 M), and effects of ovine FSH (0.037–3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2–50 ng/ml) and epidermal growth factor (EGF; 0.1–10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48–96 h and 96–144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E₂ (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P₄ output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number (∼1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E₂ (18-fold); this was accompanied by modest increases (P < 0.01) in P₄ output (∼2.5-fold) and cell number (∼2-fold). Whereas FSH enhanced inh A, act A, FS, and E₂ secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E₂. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio (∼3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin “tone” and that this could be a key requirement for FSH and IGF-induction of follicular E₂ production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E₂ output.