Hyperinsulinemic states have been associated with an increased incidence of estrogen-dependent endometrial neoplasia. To study the effect of insulin on the ability of endometrium to aromatize androgens to estrogens, late proliferative endometrium was obtained from normally cycling women at the time of indicated surgery, separated into component glands and stroma, and grown to confluence. Separated gland and stromal cultures were incubated in triplicate with increasing insulin concentrations and epidermal growth factor. Aromatase activity was assayed by the production of tritiated water from tritium-labeled androstenedione. The activity was noted to increase proportionally with increasing concentrations of insulin greater than 10 U/ml, and the effect was specific. These data suggest the following conclusions: (1) Insulin stimulates aromatase activity in both endometrial glands and stroma; (2) hyperinsulinemia may predispose to endometrial neoplasia by enhancing endogenous endometrial estrogen production.