The acute effect of epidermal growth factor (EGF) on progesterone biosynthesis by hen granulosa cells in short term culture was investigated. Pretreatment of cells for 5 h with EGF at concentrations of 1000–4000 ng/ml inhibited LHstimulated progesterone production by 54%. Shorter EGF pretreatment times of 1 and 3 h caused 25% and 35% inhibition of LH-stimulated progesterone production, respectively. In additional experiments, EGF was found to inhibit progesterone production in response to 8-bromo-cAMP (1 mM) and forskolin (100 μM) by 34% and 35%, respectively. EGF had no effect on the conversion of 25-hydroxy-cholesterol or preghenolone to progesterone, indicating that one site at which EGF inhibits progesterone biosynthesis is distal to cAMP generation, but before the side-chain cleavage step. EGF also inhibited LHstimulated cAMP production by 32%, but had no effect on forskolin-stimulated cAMP accumulation. This indicated that there was a second site of EGF action in these cells, probably at the level of LH receptor coupling to the adenylate cyclase. Nerve growth factor (4000 ng/ml) had no effect on progesterone production, but fibroblast growth factor (4000 ng/ml) facilitated LH-stimulated progesterone production. The results demonstrate that the acute inhibitory effect of EGF on LH-stimulated progesterone biosynthesis in hen granulosa cells is due to its action at two sites: One at a site before the production of cAMP and the other at a step beyond cAMP generation.).