We previously reported no difference in progesterone (P4) secretion from the granulosa layer of the largest follicle (F1) of the domestic hen regardless of the maturity of the F1 follicle. However, coincubation of the granulosa and thecal layers resulted in inhibition of P4 secretion from the less mature F1, but not from the more mature F1. The goal of this study was to determine if estradiol (E2) and androgens secreted by the thecal layer suppress P4 production by the granulosa cells. We removed the granulosa layer from less mature F1 follicles and dispersed granulosa cells (1 x 10(5)) were incubated (3 h) in triplicate with one of these treatments: control, E2, testosterone (T), androstenedione (A), and dihydrotestosterone (DHT; at concentrations of 1 x 10(-7), 1 x 10(-6), and 1 x 10(-5) M), LH (100 ng) as well as LH plus E2, T, A, and DHT at the same concentrations. P4 secretion was measured in the medium and cells, and the experiment was replicated seven times. We found a dose-related suppression of basal and LH-stimulated P4 production by all steroids. In a second experiment (n = 3-5), we tested the specificity of the androgens in suppressing P4 production by granulosa cells by using the aromatase inhibitor 7-(4'-amino)phenylthio-4-androstene-3,17-dione. This compound did not reduce the effectiveness of T in suppressing P4 production. Finally in Exp 3 (n = 4-7), E2 and T were tested individually and in combination at concentrations of 1 X 10(-8)-1 X 10(-5) M. We found a possible synergistic effect, in that the combination of E2 plus T suppressed P4 to a greater degree than either steroid alone. Our results indicate that 1) E2 and androgens suppress basal and LH-stimulated P4 production by granulosa cells in a dose-related manner; 2) androgen suppression of P4 production is not mediated by aromatization to estrogen; and 3) the suppressive effects of E2 and androgens may be synergistic. We conclude that E2 and androgens secreted by the thecal layer may regulate P4 production by the granulosa layer.