Intramyocellular lipid (IMCL), an ectopic fat depot found within skeletal muscle fibers, is highly associated with obesity and strongly correlated with insulin resistance. IMCL accumulation in sedentary individuals may contribute to insulin resistance by interfering with insulin signaling in skeletal muscle, leading to inadequate glucose uptake by the cell. Lipid peroxidation is also associated with both obesity and insulin resistance, and with IMCL, but a relationship has yet to be established among all of these variables. The purpose of this project is to study for the first time the relationships among lipid peroxidation, IMCL content, and glucose uptake in skeletal muscle. Nine insulin-sensitive adults (IS), 13 insulin-resistant adults (IR), 10 diabetic (DB) and 8 subjects pre- and post- 12-week intervention with insulin-sensitizing thiazolinedione (TZD) were assessed for soleus IMCL with nuclear magnetic resonance, insulin sensitivity by both hyperinsulinemic-euglycemic clamp (GDR) and homeostasis model assessment index (HOMA1), and anthropometrics, including body mass index (BMI), percent fat by DEXA scan, and waist circumference. Vastus lateralis biopsies of all subjects were homogenized and analyzed by immunoblotting for post-translational protein modifications occurring from lipid-peroxidation (HNE). GDR and HOMA were significantly different among IS, IR, and DB groups, as expected, as were waist circumference and BMI. IMCL was significantly higher in DB than in IS and IR. HNE was also higher in DB than in IS, although it did not differ from IR. HNE was significantly correlated to GDR, HOMA1, and BMI, but not to IMCL, WAIST, or percent fat measures. IMCL showed a strong, negative correlation with GDR and was the primary, independent predictor of GDR in stepwise multiple regression. HNE was the primary, independent predictor of HOMA in stepwise multiple regression. Paired t-tests revealed improvements in insulin sensitivity measures after 12 weeks of TZD intervention, but no significant differences were observed in IMCL or HNE after intervention. These data show that skeletal muscle HNE and IMCL are both determinants of insulin resistance in obese, sedentary adults. HNE and IMCL are not related and therefore impact insulin resistance independently. These results reveal, for the first time, a negative relationship between skeletal muscle HNE and insulin sensitivity in sedentary individuals and underscore the importance of lipid peroxidation in insulin resistance.