Endogenous opioid peptides are negative regulators of estradiol-induced uterine cell proliferation. To investigate the possible molecular target site(s) of their anti-mitogenic action, we examined the effect of opioid peptides on epidermal growth factor-induced cell proliferation both in uterine primary cell cultures prepared from adult rats and in human myometrial smooth muscle cell lines. Epidermal growth factor (EGF) significantly increased cell density in both types of cultured monolayers. This EGF-induced stimulation of cell proliferation was blocked by [D-Met(2)-Pro(5)]enkephalinamide in a time-dependent, receptor-mediated manner. The effective concentrations were within the physiological nanomolar range. Enkephalinamide did not have any effect on the basal rate of proliferation of the uterine cells. Our results on this novel physiological cross-talk suggest that shared step(s) of the mechanism of action of estradiol and EGF might be targeted by opioid peptides and not the general machinery of cell proliferation.