To investigate the respective role of nonesterified fatty acids (NEFA) oxidation and total lipid oxidation in lipid-induced insulin resistance, we measured the response of glucose metabolism to insulin in normal subjects without (control study) or with either heparin (heparin study) or triglycerides (TG) emulsion (Ivelip study) infusion. Three-step euglycemic-mild-hyperinsulinemic clamp studies were performed. Lipid and glucose metabolism were studied using indirect calorimetry and [6,6-2H2]glucose and [1-13C]palmitate infusions. NEFA concentration and turnover and oxidation rates were decreased by insulin in the control study, but were maintained during the heparin and Ivelip infusion studies. Total lipid oxidation was decreased similarly in the control and heparin studies, but was increased during the Ivelip infusion. Stimulation of glucose oxidation and utilization by insulin was reduced in the Ivelip study, but not in the heparin study. Thus, peripheral insulin resistance was observed in the presence of a combined increase in total lipid and NEFA oxidation, but not during an isolated increase in NEFA oxidation. On the other hand, insulin-induced inhibition of glucose production was impaired in both the heparin and Ivelip studies. We conclude that total lipid oxidation is a major determinant of peripheral insulin resistance, whereas hepatic insulin resistance could be induced even by a moderate increase in NEFA availability.