Prolonged elevation of plasma triglycerides and free fatty acids (FFA) reduces insulin-stimulated glucose disposal and myocellular flux through ATP synthase (fATPase). However, the early effects of lipids per se on fATPase are as yet unclear. Thus, this study examined glucose disposal and fATPase during 3 h of FFA elevation in the presence of low plasma insulinemia. Euglycemic pancreatic clamps with low-dose insulin supplementation (6 mU.m body surface area(-2).min(-1)) were performed in eight healthy men with (LIP) or without (CON) lipid infusion to measure whole body glucose disposal. (31)P/(1)H magnetic resonance spectroscopy of calf muscle was applied to quantify fATPase and concentrations of glucose 6-phosphate (G6P), inorganic phosphate (P(i)), phosphocreatine (PCr), ADP, pH, and IMCL before and during the clamps. Lipid infusion increased plasma FFA approximately twofold and decreased glucose disposal by approximately 50% (110-180 min: LIP 0.87 +/- 0.45 vs. CON 1.75 +/- 0.42 mg.kg(-1).min(-1), P = 0.002; means +/- SD). Intramyocellular G6P tended to rise only under control conditions, whereas PCr, ADP, pH, and IMCL remained unchanged from fasting in LIP and CON. Although P(i) concentrations increased by approximately 18%, fATPase remained unchanged from fasting during the clamps (LIP 10.2 +/- 2.2 vs. CON 10.5 +/- 2.6 micromol.g muscle(-1).min(-1), P = not significant). We conclude that 3 h of lipid elevation fail to affect ATP synthesis despite marked reduction of whole body glucose uptake. This suggests that lipid-induced insulin resistance results primarily from mechanisms decreasing glucose uptake rather than from direct interference of fatty acid metabolites with mitochondrial function.