Protein kinase C-ζ (PKC-ζ) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-ζ phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1^449-664) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85α subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1^449-664 by PKC-ζ induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-ζ and the classical isoforms phosphorylated rIRS-1^449-664 on Ser⁶¹². However, modification of this residue did not reduce the affinity of p85α binding to pTyr-containing peptides (amino acids 605−615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1^449-664 was then phosphorylated by PKC-ζ using [³²P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser⁴⁹⁸ and Ser⁵⁷⁰ were identified as novel phosphoserine sites targeted by PKC-ζ. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser → Ala mutants of rIRS-1^449-664. Ser⁵⁷⁰ was specifically targeted by PKC-ζ, as shown by immunoblotting with a phosphospecific antiserum against Ser⁵⁷⁰ of IRS-1. Binding of p85α to the S570A mutant was less susceptible to inhibition by PKC-ζ, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-ζ at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser⁶¹² appears not to participate in the negative control of insulin signaling, Ser⁵⁷⁰ may at least partly contribute to this process.