To explore the mechanism of MAP kinase activation in adipocytes, we examined the possible involvement of several candidate signaling proteins, MAP kinase activity was markedly increased 2-4 min after treatment with insulin and declined to basal levels after 20 min, The insulin-dependent tyrosine phosphorylation of IRS-1 in the internal membrane and its association with phosphatidylinositol 3 (PI3) kinase preceded MAP kinase activation. There was little or no tyrosine phosphorylation of She or association of Grb2 with She or IRS-1. Specific PI3 kinase inhibitors blocked the insulin-mediated activation of MAP kinase. They also decreased the activation of MAP kinase by PMA and EGF but to a much lesser extent, Insulin induced phosphorylation of AKT on serine/threonine residues, and its effect could be blocked by PI3 kinase inhibitors. These results suggest that the insulin-dependent activation of MAP kinase in adipocytes is mediated by the IRS-1/PI3 kinase pathway but not by the Shc/Grb2/SOS pathway.