We determined the effect of infusion of glucosamine (GlcN), which bypasses the rate limiting reaction in the hexosamine pathway, on insulin-stimulated rates of glucose uptake and glycogen synthesis in vivo in rat tissues varying with respect to their glutamine:fructose-6-phosphate amidotransferase (GFA) activity. Three groups of conscious fasted rats received 6-h infusions of either saline (BAS), insulin (18 mU/kg·min) and saline (INS), or insulin and GlcN (30 μmol/kg·min, GLCN). [3-³H]glucose was infused to trace whole body glucose kinetics and glycogen synthesis, and rates of tissue glucose uptake were determined using a bolus injection of[ 1-¹⁴C]2-deoxyglucose at 315 min. GlcN decreased insulin-stimulated glucose uptake (315–360 min) by 49% (P < 0.001) at the level of the whole body, and by 31–53% (P < 0.05 or less) in the heart, epididymal fat, submandibular gland and in soleus, abdominis and gastrocnemius muscles. GlcN completely abolished glycogen synthesis in the liver. GlcN decreased insulin-stimulated glucose uptake similarly in the submandibular gland (1.3 ± 0.2 vs. 2.0 ± 0.3 nmol/mg protein·min, GLCN vs. INS, P < 0.05) and gastrocnemius muscle (1.4 ± 0.3 vs. 3.1 ± 0.5 nmol/mg protein·min), although the activity of the hexosamine pathway, as judged from basal GFA activity, was 10-fold higher in the submandibular gland (286 ± 35 pmol/mg protein·min) than in gastrocnemius muscle (27 ± 3 pmol/mg protein·min, P < 0.001). These data raise the possibility that overactivity of the hexosamine pathway may contribute to glucose toxicity not only in skeletal muscle but also in other insulin sensitive tissues. They also imply that the magnitude of insulin resistance induced between tissues is determined by factors other than GFA.