We investigated the response of the glucose transport system to insulin, in the presence of ambient glucose concentrations, in isolated skeletal muscle from seven patients with non-insulin-dependent diabetes mellitus (NIDDM) (age, 55±3 years, BMI 27.4±1.8 kg/m²) and seven healthy control subjects (age, 54±3 years, BMI 26.5±1.1 kg/m²). Insulin-mediated whole body glucose utilization was similar between the groups when studied in the presence of ambient glucose concentrations (approximately 10 mmol/l for the NIDDM patients and 5 mmol/l for the control subjects). Samples were obtained from the vastus lateralis muscle, by means of an open muscle biopsy procedure, before and after a 40-min insulin infusion. An increase in serum insulin levels from 54±12 to 588±42 pmol/l, induced a 1.6±0.2-fold increase in glucose transporter protein (GLUT4) in skeletal muscle plasma membranes obtained from the control subjects (p<0.05), whereas no significant increase was noted in plasma membrane fractions prepared from NIDDM muscles, despite a similar increase in serum insulin levels. At concentrations of 5 mmol/l 3-O-methylglucose in vitro, insulin (600 pmol/l) induced a 2.2-fold (p<0.05) increase in glucose transport in NIDDM muscles and a 3.4-fold (p<0.001) increase in the control muscles. Insulin-stimulated 3-O-methylglucose transport was positively correlated with whole body insulin-mediated glucose uptake in all participants (r=0.78,p<0.001) and negatively correlated with fasting plasma glucose levels in the NIDDM subjects (r=0.93,p<0.001). Muscle fibre type distribution and capillarization were similar between the groups. Our results suggest that insulin-stimulated glucose transport in skeletal muscle from patients with NIDDM is down-regulated in the presence of hyperglycaemia. The increased flux of glucose as a consequence of hyperglycaemia may result in resistance to any further insulin-induced gain of GLUT4 at the level of the plasma membrane.