Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at serine³³². However, the fact that GSK-3 requires pre-phosphorylation of its substrates suggested that serine³³⁶ on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at serines 336 and 332 in physiologically relevant animal models. Of several stimulators, only the PKC activator, phorbol-ester PMA, enhanced IRS-1 phosphorylation at serine336. Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKC or PKCII isoforms in cells enhanced IRS-1 phosphorylation at serines 336 and 332 and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at serine³³⁶. The expression level and activation state of PKCII but not PKC, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat-diet-fed mice as compared with that from lean animals. Elevated levels of PKCII were also associated with enhanced phosphorylation of IRS-1 at serines^336/332 and elevated activity of GSK-3. Finally, adenoviral-mediated expression of PKC II in adipocytes enhanced phosphorylation of IRS-1 at serine³³⁶.Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCII and GSK-3 at serines 336 and 332. Further, this data provides evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in the fat tissue.