Otsuka Long-Evans Tokushima Fatty (OLETF) rats showed that the distribution of plasma membrane content of insulin-regulated glucose transporter in skeletal muscle was reminiscent of that in human non-insulin-dependent diabetes mellitus (NIDDM). To obtain more information on the cellular mechanisms of muscle insulin resistance, hexokinase activities were measured in the skeletal muscle of OLETF rats. The results showed that the activity of the type II enzyme in the diabetic rats was significantly decreased (P < 0.05) compared with Long-Evans Tokushima Otsuka (LETO) control rats. No significant differences in the activity of the type I hexokinase were observed between these rats. Western blot analysis showed that the protein content of the type II in OLETF rats was also significantly lower than that in LETO rats (P < 0.05). After insulin stimulation, the intramuscular content of glucose 6-phosphate, which regulates glycogen synthesis in skeletal muscle, was significantly decreased in OLETF rats (P < 0.01). However, glycogen synthase activity in vitro and intramuscular lactate concentration in these rats did not show significant differences. These results suggest that the G6P content of the diabetic rats is decreased as a result of an impaired early event of glucose metabolism, indicating that the molecular defects of skeletal muscle in OLETF rats are similar to those in NIDDM patients.