The impact of long-term lithium exposure on the replication, polyamine content, and insulin production of rat pancreatic beta-cells was examined. Fetal rat pancreatic islets enriched in beta-cells were isolated and cultured for 3 days in the presence of different concentrations of LiCl. It was found that lithium dose dependently stimulated beta-cell replication, evoking a 40% increase in beta-cell replication at 1 mM, which was further increased to 70% at 10 mM of the ion. In contrast, the islet contents of insulin mRNA and insulin and insulin biosynthesis rates remained unaltered. The long-term insulin accumulation in the culture medium was nevertheless increased in LiCl-treated groups. In addition, neither the mitogenic effect nor the increased insulin accumulation in the medium by lithium was further augmented by growth hormone, which itself stimulated these functions. Lithium was also found to elevate the islet content of polyamines. Treatment with enzymatic inhibitors of polyamine synthesis failed to preclude the mitogenic and secretagogic impact of lithium, suggesting that the increased contents of polyamines did not convey the lithium effect. We conclude that lithium treatment stimulates rat beta-cell replication and long-term insulin secretion in vitro. These direct effects of the ion on the beta-cell may contribute to the antidiabetic effect of lithium encountered in animal models and patients.