Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 approximately 6 microM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.