In this study, luteinized human granulosa cells (GC) obtained during in vitro fertilization procedures were used as a model system to evaluate the effects of ethanol (EtOH), a well-known reproductive toxin, on epidermal growth factor (EGF) and gonadotropin-stimulated steroidogenesis. Our results demonstrate that the basal progesterone (P4) and estradiol (E2) secretion by human GC in vitro was dependent on the ovarian stimulation protocol. EGF significantly enhanced P4, but not E2, secretion in human GC from clomiphene citrate (CC), human menopausal gonadotropin (hMG), and hMG/gonadotropin-releasing hormone agonist (GnRH-a)-treated patients. The effects of EGF plus luteinizing hormone (LH) were additive in cells from the CC group, but less than additive in hMG and hMG/GnRH-a groups. EtOH at 20 mM or more inhibited EGF stimulated P4 secretion in human GC from all three patient groups. EtOH inhibited P4 secretion stimulated by EGF and LH cotreatment in the CC and hMG/GnRH-a groups, but not in human GC from the hMG-treated patients. These results suggest that basal and EGF or LH-stimulated P4 secretion by human GC, as well as the effects of EtOH, are profoundly influenced by the follicle's hormonal milieu.