This study investigates the mechanism by which free fatty acids (FFA) induce insulin resistance in humans. Nine healthy subjects underwent euglycemic-hyperinsulinemic clamps with low or high plasma FFA levels. During the first 3.5 hours, whole-body glucose uptake was not affected by lipid infusion, but it decreased continuously to about 46% of control values after 6 hours. Augmented lipid oxidation was accompanied by a 40% reduction in oxidative glucose metabolism starting in the third hour. Muscle glycogen synthesis rates were similar during the first 3 hours but decreased to about 50% of control values after that. The reduction in muscle glycogen synthesis was preceded by a fall in muscle glucose-6-phosphate concentrations starting at about 1.5 hours. These results demonstrate that free fatty acids induce insulin resistance in humans by initially inhibiting glucose transport/phosphorylation, followed by a 50% reduction in both muscle glycogen synthesis and glucose oxidation. The study challenges the previously proposed mechanism in which FFA inhibit insulin-stimulated glucose uptake through initial inhibition of pyruvate dehydrogenase. Instead, the findings suggest that FFA inhibit glucose transport and phosphorylation, leading to reduced glucose oxidation and glycogen synthesis. This mechanism is consistent with the observed reduction in intramuscular glucose-6-phosphate concentrations during lipid infusion. The study also highlights the importance of intramuscular FFA metabolism in the pathogenesis of insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). The results suggest that alterations in intramuscular FFA metabolism may play a significant role in the development of insulin resistance. The study was supported by grants from the Public Health Service and the American Diabetes Association.This study investigates the mechanism by which free fatty acids (FFA) induce insulin resistance in humans. Nine healthy subjects underwent euglycemic-hyperinsulinemic clamps with low or high plasma FFA levels. During the first 3.5 hours, whole-body glucose uptake was not affected by lipid infusion, but it decreased continuously to about 46% of control values after 6 hours. Augmented lipid oxidation was accompanied by a 40% reduction in oxidative glucose metabolism starting in the third hour. Muscle glycogen synthesis rates were similar during the first 3 hours but decreased to about 50% of control values after that. The reduction in muscle glycogen synthesis was preceded by a fall in muscle glucose-6-phosphate concentrations starting at about 1.5 hours. These results demonstrate that free fatty acids induce insulin resistance in humans by initially inhibiting glucose transport/phosphorylation, followed by a 50% reduction in both muscle glycogen synthesis and glucose oxidation. The study challenges the previously proposed mechanism in which FFA inhibit insulin-stimulated glucose uptake through initial inhibition of pyruvate dehydrogenase. Instead, the findings suggest that FFA inhibit glucose transport and phosphorylation, leading to reduced glucose oxidation and glycogen synthesis. This mechanism is consistent with the observed reduction in intramuscular glucose-6-phosphate concentrations during lipid infusion. The study also highlights the importance of intramuscular FFA metabolism in the pathogenesis of insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). The results suggest that alterations in intramuscular FFA metabolism may play a significant role in the development of insulin resistance. The study was supported by grants from the Public Health Service and the American Diabetes Association.