Effects of 24-h and 48-h fasting on maximal insulin-stimulated whole-body and muscle glucose uptake, glycogen synthesis, and glycolysis were studied in conscious rats by combining the glucose clamp technique with tracer methods. Fasting decreased body weight and basal plasma glucose, plasma insulin, hepatic glucose output, and glucose clearance (P < 0.05 for all). However, maximal insulin-stimulated whole-body glucose uptake, normalized to body weight, was almost identical in fed, 24-h fasted, and 48-h fasted rats (191 ± 8, 185 ± 14, and 182 ± 5 μmol · kg⁻¹ · min⁻¹, respectively; P > 0.7). Similarly, rates of insulin-stimulated glucose uptake by four different skeletal muscles, estimated by the 2-deoxyglucose injection technique, were not different among the three groups. In contrast to glucose uptake, insulin-stimulated whole-body glycolysis was decreased significantly after fasting (36% after 48 h fasting; P < 0.05), whereas insulin-stimulated whole-body glycogen synthesis was increased (44% after 48 h fasting; P < 0.05). In fed rats, glycolysis was the major pathway for glucose metabolism during hyperinsulinemia, accounting for 60 ± 5% of glucose uptake. This fraction was decreased significantly by fasting (P < 0.01), so that after a 48-h fast, glycolysis accounted for only 40 ± 3% of insulin-stimulated glucose uptake and glycogen synthesis became predominant pathway, accounting for 60 ± 3% of whole-body glucose utilization. Whole-body patterns of glucose metabolism during hyperinsulinemia were paralleled by glucose metabolism in individual muscles. These data indicate that fasting for up to 48 h in rats had no effect on insulin-stimulated glucose disposal (whole body or muscle) but resulted in a change of the primary metabolic path for insulin-stimulated glucose utilization from glycolysis to glycogen synthesis.