In this study, we demonstrate that stimulation of beta cells with carbachol and glucose causes increased tyrosine phosphorylation of a 125-kDa protein concurrently with increased insulin secretion. The effect was observed in two different insulin-secreting cell lines and in rat pancreatic islets. Tyrosine phosphorylation was largely calcium independent and occurred within 2 min after stimulation of beta cells with glucose and the muscarinic agonist carbachol. In islets, the effect of glucose was greatly diminished by the addition of mannoheptulose, a seven-carbon sugar that inhibits glucokinase, suggesting that glucose metabolism is required for tyrosine phosphorylation of the protein to occur. Neither insulin nor insulin-like growth factor I significantly increased tyrosine phosphorylation of the 125-kDa protein, suggesting that it was not an autocrine effect. Depolarization of beta cells with glyburide or 50 m potassium dramatically increased insulin secretion but had no significant effect on tyrosine phosphorylation. Addition of phorbol ester caused a less than 2-fold increase in tyrosine phosphorylation, whereas the calcium ionophore A23187 had no effect. Among the various fuel secretagogues tested, only -glucose stimulated tyrosine phosphorylation, both alone and in combination with carbachol. Finally, the tyrosine kinase inhibitor AG879 inhibited both tyrosine phosphorylation and insulin secretion in a dose-dependent manner. Taken together, these data demonstrate the presence of a novel signaling pathway in glucose-induced insulin secretion: tyrosine phosphorylation of beta cell p125, which is a proximal step in insulin secretion. Our current working hypothesis is that glucose stimulation of beta cell p125 tyrosine phosphorylation is an essential step for insulin secretion.