Abstract Skeletal muscle contributes significantly to reduced insulin-stimulated glucose disposal in patients with obesity and non-insulin-dependent (type II) diabetes mellitus (NIDDM). The biochemical basis for insulin resistance is not known but may involve reduced glucose transport and/or a defect in intracellular pathways for glucose disposal. To address this question, we measured basal and insulin-stimulated glucose oxidation, glycogen formation, and nonoxidative glycolysis (lactate and amino acid release) in an incubated muscle preparation from nonobese and morbidly obese patients with and without NIDDM. Pathways of glucose disposal were also determined in muscle of obese NIDDM patients incubated under hyperglycemic (20 mmol/L) conditions, which increases glucose uptake by mass action. Under basal conditions (no insulin present) there were no significant differences in glycogen formation or glucose oxidation between nonobese control, obese nondiabetic, or obese diabetics. Lactate release was significantly higher in obese controls compared to nonobese controls in the basal state at 5 mmol/L glucose (10.2 ± 2.8 v 24.7 ± 3.5 nmol/min/g, P < .05). Under maximal insulin-stimulated conditions, rates of glycogen formation, glucose oxidation, and nonoxidized glycolysis increased 1.9-, 2.3-, and 2.2-fold over basal ( P < .05) in nonobese controls. By contrast, insulin was ineffective at stimulating significant increases in any metabolic pathway of glucose disposal in muscle of obese or obese NIDDM patients. When muscle of obese diabetics was incubated at 20 mmol/L glucose however, insulin responsiveness (maximal rate minus basal rate) increased significantly ( P < .05) for glycogen formation, glucose oxidation, and nonoxidative glycolysis (4.50 ± 1.46, 0.53 ± 0.18, 19.9 ± 5.6 nmol/min/g, respectively), to levels similar to nonobese controls (3.61 ± 1.06, 0.32 ± 0.13, 12.5 ± 3.5 nmol/min/g). These data indicate that all pathways of insulin-stimulated glucose disposal are reduced in incubated muscle of morbidly obese and obese diabetics. However, hyperglycemia increases basal glucose transport and increases insulin responsiveness of glucose disposal in incubated human muscle.