Protein kinase C-ζ, a downstream effector of phosphatidylinositol 3-kinase (PI3K), phosphorylates insulin receptor substrate (IRS)-1 on serine residues impairing activation of PI3K in response to insulin. Because IRS-1 is upstream from PI3K, this represents a negative feedback mechanism that may contribute to signal specificity in insulin action. To determine whether similar feedback pathways exist for other IRS isoforms, we evaluated IRS-2, -3, and -4 as substrates for PKC-ζ. In an in vitro kinase assay, purified recombinant PKC-ζ 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-ζ, phosphorylated IRS-1, -3, and -4 but not IRS-2. We evaluated functional consequences of serine phosphorylation of IRS isoforms by PKC-ζ in NIH-3T3IR cells cotransfected with epitope-tagged IRS proteins and either PKC-ζ or empty vector control. Insulin-stimulated IRS tyrosine phosphorylation was impaired by overepxression of PKC-ζ for IRS-1, -3, and -4 but not IRS-2. Significant insulin-stimulated increases in PI3K activity was coimmunoprecipitated with all IRS isoforms. In cells overexpressing PKC-ζ 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-ζ. We conclude that IRS-3 and -4 are novel substrates for PKC-ζ that may participate in a negative feedback pathway for insulin signaling similar to IRS-1. The inability of PKC-ζ to phosphorylate IRS-2 may help determine specific functional roles for IRS-2. Copyright © 2008 by The Endocrine Society.