We investigated the influence of altered glucose levels on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from NIDDM patients (n = 13) and non-diabetic subjects (n = 23). Whole body insulin sensitivity was 71% lower in the NIDDM patients compared to the non-diabetic subjects (p < 0.05), whereas, insulin-mediated peripheral glucose utilization in the NIDDM patients under hyperglycaemic conditions was comparable to that of the non-diabetic subjects at euglycaemia. Following a 30-min in vitro exposure to 4 mmol/l glucose, insulin-stimulated 3-0-methylglucose transport (600 pmol/l insulin) was 40% lower in isolated skeletal muscle strips from the NIDDM patients when compared to muscle strips from the non-diabetic subjects. The impaired capacity for insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle was normalized following prolonged (2h) exposure to 4 mmol/l, but not to 8 mmol/l glucose. Insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle exposed to 8 mmol/l glucose was similar to that of the non-diabetic muscle exposed to 5 mmol/l glucose, but was decreased by 43% (p < 0.01) when compared to non-diabetic muscle exposed to 8 mmol/l glucose. Despite the impaired insulin-stimulated 3-0-methylglucose transport capacity demonstrated by skeletal muscle from the NIDDM patients, skeletal muscle glycogen content was similar to that of the non-diabetic subjects. Kinetic studies revel a Km for 3-0-methylglucose transport of 9.7 and 8.8 mmol/l glucose for basal and insulin-stimulated conditions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)