Insulin resistance for glucose metabolism in skeletal muscle is a key feature in NIDDM. The quantitative role of the cellular effectors of glucose metabolism in determining this insulin resistance is still imperfectly known. We assessed transmembrane glucose transport and intraceUular glucose phosphorylation in vivo in skeletal muscle in nonobese NIDDM patients. We performed euglycemic insulin clamp studies in combination with the forearm balance technique (brachial artery and deep forearm vein catheterization) in five nonobese NIDDM patients and seven age- and weight-matched control subjects (study 1). d-Mannitol (a nontransportable molecule), 3-O-[¹⁴C]methyl-d-glucose (transportable, but not metabolizable) and d[3–³H]glucose (transportable and metabolizable) were simultaneously injected into the brachial artery, and the washout curves were measured in the deep venous effluent blood. In vivo rates of transmembrane transport and intracellular phosphorylation of d-glucose in forearm muscle were determined by analyzing the washout curves with the aid of a multicompartmental model of glucose kinetics in forearm tissues. At similar steady-state concentrations of plasma insulin (∼500 pmol/l) and glucose (∼5.0 mmol/l), the rates of transmembrane influx (34.3 ± 9.1 vs. 58.5 ± 6.5 μmol · min^-1 μ kg^-1 P < 0.05) and intracellular phosphorylation (5.4 ± 1.6 vs. 38.8 ± 5.1 μmol μ min^-1 · kg^-1 P < 0.01) in skeletal muscle were markedly lower in the NIDDM patients than in the control subjects. In the NIDDM patients (study 2), the insulin clamp was repeated at hyperglycemia (∼13 mmol/l) trying to match the rates of transmembrane glucose influx measured during the clamp in the controls. The rate of transmembrane glucose influx (62 ± 15 μmol · min^-1 · kg^-1) in the NIDDM patients was similar to the control subjects, but the rate of intracellular glucose phosphorylation (16.6 ± 7.5 μmol · min^-1 · kg^-1), although threefold higher than in the patients during study 1 (P < 0.05), was still ∼60% lower than in the control subjects (P < 0.05). These data suggest that when assessed in vivo, both transmembrane transport and intracellular phosphorylation of glucose are refractory to insulin action and add to each other in determining insulin resistance in skeletal muscle of NIDDM patients. It will be of interest to compare the present results with the in vivo quantitation of the initial rate of muscle glucose transport when methodology to perform this measurement becomes available.