Overactivity of the hexosamine pathway mediates glucose-induced insulin resistance in rat adipocytes. Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme of this pathway. We determined GFA activity in human skeletal muscle biopsies and rates of insulin-stimulated whole-body, oxidative, and nonoxidative glucose disposal using the euglycemic insulin clamp technique combined with indirect calorimetry (insulin infusion rate (1.5 mU x kg-1 x min-1)) in 12 male patients with NIDDM (age 54 +/- 2 years, BMI 27.5 +/- 0.9 kg/m2, fasting plasma glucose 8.5 +/- 0.6 mmol/l) and 9 matched normal men. GFA activity was detectable in human skeletal muscles and completely inhibited by uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) in all subjects. GFA activity was 46% increased in the NIDDM patients compared with the normal subjects (9.5 +/- 1.3 vs. 6.5 +/- 1.2 pmol, P < 0.05). Whole-body glucose uptake was 58% decreased in patients with NIDDM (20 +/- 3 micromol x kg body wt-1 x min-1) compared with normal subjects (47 +/- 4 micromol x kg body wt-1 x min-1, P < 0.001). This decrease was attributable to decreases in both glucose oxidation (9 +/- 1 vs. 15 +/- 1 micromol x kg-1 x min-1, NIDDM patients vs. control subjects, P < 0.002) and nonoxidative glucose disposal (11 +/- 2 vs. 31 +/- 4 micromol x kg-1 x min-1, P < 0.001). In patients with NIDDM, both HbA1c (r= 0.51, P < 0.05) and BMI (r= -0.57, P < 0.05) correlated with whole-body glucose uptake. HbA1c but not BMI or insulin sensitivity was correlated with basal GFA activity (r = -0.57,P < 0.01) in NIDDM patients and control subjects. We conclude that GFA is found in human skeletal muscle and that all this activity is sensitive to feedback inhibition by UDP-GlcNAc. Chronic hyperglycemia is associated with an increase in skeletal muscle GFA activity, suggesting that increased activity of the hexosamine pathway may contribute to glucose toxicity and insulin resistance in humans.