After entering the muscle cell, glucose is immediately and irreversibly phosphorylated to glucose-6-phosphate by hexokinases (HK) I and II. Previous studies in rodents have shown that HKII may be the dominant HK in skeletal muscle. Reduced insulin-stimulated glucose uptake and reduced glucose-6-phosphate concentrations in muscle have been found in non-insulin-dependent diabetes mellitus (NIDDM) patients when examined during a hyperglycemic hyperinsulinemic clamp. These findings [correction of finding] are consistent with a defect in glucose transport and/or phosphorylation. In the present study comprising 29 NIDDM patients and 25 matched controls, we tested the hypothesis that HKII activity and gene expression are impaired in vastus lateralis muscle of NIDDM patients when examined in the fasting state. HKII activity in a supernatant of muscle extract accounted for 28 +/- 5% in NIDDM patients and 40 +/- 5% in controls (P = 0.08) of total muscle HK activity when measured at a glucose media of 0.11 mmol/liter and 31 +/- 4 and 47 +/- 7% (P = 0.02) when measured at 0.11 mmol/liter of glucose. HKII mRNA, HKII immunoreactive protein level, and HKII activity were significantly decreased in NIDDM patients (P < 0.0001, P = 0.03, and P = 0.02, respectively) together with significantly decreased glycogen synthase mRNA level and total glycogen synthase activity (P = 0.02 and P = 0.02, respectively). In the entire study population HKII activity estimated at 0.11 and 11.0 mM glucose was inversely correlated with fasting plasma glucose concentrations (r = -0.45, P = 0.004; r = -0.54, P < 0.0001, respectively) and fasting plasma nonesterified fatty acid concentrations (r = -0.46, P = 0.003; r = -0.37, P = 0.02, respectively). In conclusion, NIDDM patients are characterized by a reduced activity and a reduced gene expression of HKII in muscle which may be secondary to the metabolic peturbations. HKII contributes with about one-third of total HK activity in a supernatant of human vastus lateralis muscle.