Protein Kinase C- (PKC-), a downstream effector of phosphatidylinositol 3-kinase (PI3K), phosphorylates insulin receptor substrate 1 (IRS-1) on serine residues impairing activation of PI3K in response to insulin. Since IRS-1 is upstream from PI3K, this represents a negative feedback mechanism that may contribute to signal specificity in insulin action. To determine if similar feedback pathways exist for other IRS isoforms, we evaluated IRS-2, -3, and -4 as substrates for PKC-. In an in vitro kinase assay, purified recombinant PKC- phosphorylated IRS-1, -3 and -4, but not IRS-2. Similar results were obtained with an immune-complex kinase assay demonstrating that wild-type, but not kinase-deficient mutant PKC-, phosphorylated IRS-1, -3, and -4, but not IRS-2. We evaluated functional consequences of serine phosphorylation of IRS isoforms by PKC- in NIH-3T3^IR cells co-transfected with epitope-tagged IRS proteins and either PKC- or empty vector control. Insulin-stimulated IRS tyrosine phosphorylation was impaired by overepxression of PKC- for IRS-1, -3, and -4, but not IRS-2. Significant insulin-stimulated increases in PI3K activity was co-immunoprecipitated with all IRS isoforms. In cells overexpressing PKC- there was marked inhibition of insulin-stimulated PI3K activity associated with IRS-1, -3 and -4, but not IRS-2. That is, PI3K activity associated with IRS-2 in response to insulin was similar in control cells and cells overexpressing PKC-. We conclude that IRS-3 and -4 are novel substrates for PKC- that may participate in a negative feedback pathway for insulin signaling similar to IRS-1. The inability of PKC- to phosphorylate IRS-2 may help determine specific functional roles for IRS-2.