To examine whether the hexosamine biosynthetic pathway might play a role in fat-induced insulin resistance, we monitored the effects of prolonged elevations in FFA availability both on skeletal muscle levels of UDP-N-acetyl-hexosamines and on peripheral glucose disposal during 7-h euglycemic-hyperinsulinemic (approximately 500 microU/ml) clamp studies. When the insulin-induced decrease in the plasma FFA levels (to approximately 0.3 mM) was prevented by infusion of a lipid emulsion in 15 conscious rats (plasma FFA approximately 1.4 mM), glucose uptake (5-7 h = 32.5+/-1.7 vs 0-2 h = 45.2+/-2.8 mg/kg per min; P < 0.01) and glycogen synthesis (P < 0.01) were markedly decreased. During lipid infusion, muscle UDP-N-acetyl-glucosamine (UDP-GlcNAc) increased by twofold (to 53.4+/-1.1 at 3 h and to 55.5+/-1.1 nmol/gram at 7 h vs 20.4+/-1.7 at 0 h, P < 0.01) while glucose-6-phosphate (Glc-6-P) levels were increased at 3 h (475+/-49 nmol/gram) and decreased at 7 h (133+/-7 vs 337+/-28 nmol/gram at 0 h, P < 0.01). To discern whether such an increase in the skeletal muscle UDP-GlcNAc concentration could account for the development of insulin resistance, we generated similar increases in muscle UDP-GlcNAc using three alternate experimental approaches. Euglycemic clamps were performed after prolonged hyperglycemia (18 mM, n = 10), or increased availability of either glucosamine (3 micromol/kg per min; n = 10) or uridine (30 micromol/kg per min; n = 4). These conditions all resulted in very similar increases in the skeletal muscle UDP-GlcNAc (to approximately 55 nmol/gram) and markedly impaired glucose uptake and glycogen synthesis. Thus, fat-induced insulin resistance is associated with: (a) decreased skeletal muscle Glc-6-P levels indicating defective transport/phosphorylation of glucose; (b) marked accumulation of the endproducts of the hexosamine biosynthetic pathway preceding the onset of insulin resistance. Most important, the same degree of insulin resistance can be reproduced in the absence of increased FFA availability by a similar increase in skeletal muscle UDP-N-acetyl-hexosamines. In conclusion, our results support the hypothesis that increased FFA availability induces skeletal muscle insulin resistance by increasing the flux of fructose-6-phosphate into the hexosamine pathway.