During pregnancy, pancreatic islets undergo structural and functional changes in response to an increased demand for insulin. Different hormones, especially placental lactogens, mediate these adaptive changes. Prolactin (PRL) mainly exerts its biological effects by activation of the JAK2/STAT5 pathway. PRL also stimulates some biological effects via activation of IRS-1, IRS-2, PI 3-kinase, and MAPK in different cell lines. Since IRS-2 is important for the maintenance of pancreatic islet cell mass, we investigated whether PRL affects insulin-signaling pathways in neonatal rat islets. PRL significantly potentiated glucose-induced insulin secretion in islets cultured for 7 days. This effect was blocked by the specific PI 3-kinase inhibitor wortmannin. To determine possible effects of PRL on insulin-signaling pathways, fresh islets were incubated with or without the hormone for 5 or 15 min. Immunoprecipitation and immunoblotting with specific antibodies showed that PRL induced a dose-dependent IRS-1 and IRS-2 phosphorylation compared to control islets. PRL-induced increase in IRS-1/-2 phosphorylation was accompanied by an increase in the association with and activation of PI 3-kinase. PRL-induced IRS-2 phosphorylation and its association with PI 3-kinase did not add to the effect of insulin. PRL also induced JAK2, SHC, ERK1 and ERK2 phosphorylation in neonatal islets, demonstrating that PRL can activate MAPK. These data indicate that PRL can stimulate the IRSs/PI 3-kinase and SHC/ERK pathways in islets from neonatal rats.