A relative decrease in beta-cell mass is key in the pathogenesis of type 1 diabetes, type 2 diabetes, and in the failure of transplanted islet grafts. It is now clear that beta-cell duplication plays a dominant role in the regulation of adult beta-cell mass. Therefore, knowledge of the endogenous regulators of beta-cell replication is critical for understanding the physiological control of beta-cell mass and for harnessing this process therapeutically. We have shown that concentrations of insulin known to exist in vivo act directly on beta-cells to promote survival. Whether insulin stimulates adult beta-cell proliferation remains unclear. We tested this hypothesis using dispersed primary mouse islet cells double labeled with 5-bromo-2-deoxyuridine and insulin antisera. Treating cells with 200-pm insulin significantly increased proliferation from a baseline rate of 0.15% per day. Elevating glucose from 5-15 mm did not significantly increase beta-cell replication. beta-Cell proliferation was inhibited by somatostatin as well as inhibitors of insulin signaling. Interestingly, inhibiting Raf-1 kinase blocked proliferation stimulated by low, but not high (superphysiological), insulin doses. Insulin-stimulated mouse insulinoma cell proliferation was dependent on both phosphatidylinositol 3-kinase/Akt and Raf-1/MAPK kinase pathways. Overexpression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of 200-pm insulin. Together, these results demonstrate for the first time that insulin, at levels that have been measured in vivo, can directly stimulate beta-cell proliferation and that Raf-1 kinase is involved in this process. These findings have significant implications for the understanding of the regulation of beta-cell mass in both the hyperinsulinemic and insulin-deficient states that occur in the various forms of diabetes.