Insulin receptor (IR) may play an essential role in the development of beta-cell mass in the mouse pancreas. To further define the function of this signaling system in beta-cell development, we generated IR-deficient beta-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing beta-cell lines (beta-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (beta-IR(-/-)). beta-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In beta-IRLoxP beta-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S(6)K phosphorylation and beta-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S(6)K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S(6)K signaling and mitogenesis. Interestingly, in beta-IR(-/-) beta-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S(6)K phosphorylation and beta-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S(6)K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of beta-IR(-/-) cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic beta-cell lines. However, in the absence of the insulin receptor, glucose induces beta-cell mitogenesis.