Myoblasts from human skeletal muscle were isolated from needle biopsy samples of vastus lateralis and fused to differentiated multinucleated myotubes. Specific high-affinity insulin and insulin-like growth factor I (IGF-I) binding, glucose transporter proteins GLUT1 and GLUT4, glycogen synthase and pyruvate dehydrogenase proteins, and their specific mRNAs were identified in fused myotubes. Insulin and IGF-I stimulated 2-deoxyglucose uptake twofold with half-maximal stimulation by insulin at 0.98 ± 0.12 nmol/l and maximal stimulation at 17.5 nmol/l. Acute insulin treatment (33 nmol/1) doubled glycogen synthase activity and glucose incorporation into glycogen while increasing pyruvate dehydrogenase ∼30%. In cells cultured from NIDDM subjects, both basal (6.9 ± 1.0 vs. 13.0 ± 1.7 pmol · mg protein⁻¹ · min⁻¹) and acute insulin-stimulated transport (13.5 ± 2.0 vs. 22.4 ± 1.3 pmol · mg protein⁻¹ · min^–1) were significantly reduced compared with nondiabetic control subjects (both P ≤ 0.005). GLUT1 protein content of total membranes from NIDDM subjects was decreased compared with control subjects, while GLUT4 levels were similar between groups. A significant correlation (r = 0.65, P ≤ 0.05) was present when maximal rates of insulin-stimulated glucose transport in cell culture from subjects were compared with their corresponding in vivo glucose disposal determined by hyperinsulinemic glucose clamp. In summary, differentiated human skeletal muscle cultures exhibit biochemical and molecular features of insulin-stimulated glucose transport and intracellular enzyme activity comparable with the in vivo situation. Defective insulin-stimulated glucose transport persists in muscle cultures from NIDDM subjects and resembles the reduced insulin-mediated glucose uptake present in vivo. We conclude that this technique provides a relevant cellular model to study insulin action and glucose metabolism in normal subjects and determine the mechanisms of insulin resistance in NIDDM.