To examine the mechanisms of hyperglycemia-induced insulin resistance, eight insulin-dependent (type I) diabetic men were studied twice, after 24 h of hyperglycemia (mean blood glucose 20.0 ± 0.3 mM, i.v. glucose) and after 24 h of normoglycemia (7.1 ± 0.4 mM, saline) while receiving identical diets and insulin doses. Whole-body and forearm glucose uptake were determined during a 300-min insulin infusion (serum free insulin 359 ± 22 and 373 ± 29 pM, after hyper- and normoglycemia, respectively). Muscle biopsies were taken before and at the end of the 300-min insulin infusion. Plasma glucose levels were maintained constant during the 300-min period by keeping glucose for 150 min at 16.7 ± 0.1 mM after 24-h hyperglycemia and increasing it to 16.5 ± 0.1 mM after normoglycemia and by allowing it thereafter to decrease in both studies to normoglycemia. During the normoglycemic period (240–300 min), total glucose uptake (25.0 ± 2.8 vs. 33.8 ± 3.9 μmol·kg⁻¹ body wt·min⁻¹ P < 0.05) was 26% lower, forearm glucose uptake (11 ± 4 vs. 18 ± 3 μmol.kg⁻¹ forearm.min⁻¹ P < 0.05) was 35% lower, and nonoxidative glucose disposal (8.9 ± 2.2 vs. 19.4 ± 3.3 μmol.kg⁻¹ body wt⁻¹min⁻¹, P < 0.01) was 54% lower after 24 h of hyper- and normoglycemia, respectively. Glucose oxidation rates were similar. Basal muscle glycogen content was similar after 24 h of hyperglycemia (234 ± 23 mmol/kg dry muscle) and normoglycemia (238 ± 22 mmol/kg dry muscle). Insulin increased muscle glycogen to 273 ± 22 mmol/kg dry muscle after 24 h of hyperglycemia and to 296 ± 33 mmol/kg dry muscle after normoglycemia (P < 0.05 vs. 0 min for both). Muscle ATP, free glucose, glucose-6-phosphate, and fructose-6-phosphate concentrations were similar after both 24-h treatment periods and did not change in response to insulin. We conclude that a marked decrease in whole-body, muscle, and nonoxidative glucose disposal can be induced by hyperglycemia alone.