Signal transduction from the insulin receptor to downstream effectors is attenuated by phosphorylation at a number of Ser/Thr residues of insulin receptor substrate-1 (IRS-1) resulting in resistance to insulin action, the hallmark of type II diabetes. Ser/Thr residues can also be reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) monosaccharide, a dynamic posttranslational modification that offers an alternative means of protein regulation to phosphorylation. To identify sites of O-GlcNAc modification in IRS-1, recombinant rat IRS-1 isolated from HEK293 cells was analyzed by two complementary mass spectrometric methods. Using data-dependent neutral loss MS3 mass spectrometry, MS/MS data were scanned for peptides that exhibited a neutral loss corresponding to the mass of N-acetylglucosamine upon dissociation in an ion trap. This methodology provided sequence coverage of 84% of the protein, permitted identification of a novel site of phosphorylation at Thr-1045, and facilitated the detection of an O-GlcNAc-modified peptide of IRS-1 at residues 1027-1073. The level of O-GlcNAc modification of this peptide increased when cells were grown under conditions of high glucose with or without chronic insulin stimulation or in the presence of an inhibitor of the O-GlcNAcase enzyme. To map the exact site of O-GlcNAc modification, IRS-1 peptides were chemically derivatized with dithiothreitol following beta-elimination and Michael addition prior to LC-MS/MS. This approach revealed Ser-1036 as the site of O-GlcNAc modification. Site-directed mutagenesis and Western blotting with an anti-O-GlcNAc antibody suggested that Ser-1036 is the major site of O-GlcNAc modification of IRS-1. Identification of this site will facilitate exploring the biological significance of the O-GlcNAc modification.