Influences of estrogen and progesterone on the development of hyperinsulinemia and augmented pancreatic islet insulin secretion during pregnancy were assessed in this study. Groups of female rats were injected subcutaneously for 21 days with varying daily dosages of estradiol benzoate or progesterone in oil. On day 21, pancreatic islets were isolated by a collagenase method. Total insulin secretion was measured after 90-min incubations of 10 islets in buffered medium containing glucose. Higher physiologic dosages of estradiol or progesterone, singly or in combination, significantly increased islet secretion above values of untreated control rats and were comparable to augmented islet responses of term, 3-wk pregnant rats. Diameter and protein content of islets obtained from steroid-treated and pregnant rats exceeded control measurements in these instances. However, 2-hr preincubations of control islets with 1 or 10 mug/ml of either steroid did not influence subsequent glucose-stimulated insulin output. In related studies, plasma insulin responses during 30 min intravenous glucose tolerance tests were significantly above control responses in term-pregnant rats and animals receiving comparable dosages of steroids for 3 wk. Unlike pregnancy or progesterone treatment, estradiol administration alone or with progesterone significantly lowered postchallenge plasma glucose concentrations. These results indicate that estradiol and progesterone contribute to enhanced islet insulin secretion and plasma insulin responses to glucose administration during pregnancy. This change is not acutely produced but can be related to hypertrophy of islets following chronic hormonal administration. Although the data do not distinguish between direct and indirect beta-cytotrophic effects of these sex steroids, metabolic actions of estradiol and progesterone may differ, since estrogen treatment lowers plasma glucose curves following the induction of hyperinsulinemia.