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A rterial concentrations and net substrate exchange across the leg and splanchnic vascular bed were determined for glucose, lactate, pyruvate, and glycerol in healthy postabsorptive subjects at rest and during 40 min of exercise on a bicycle ergometer at work intensities of 400, 800, and 1200 kg-m/min.Rising arterial glucose levels and small decreases in plasma insulin concentrations were found during heavy exercise. Significant arterial-femoral venous differences for glucose were demonstrated both at rest and during exercise, their magnitude increasing with work intensity as well as duration of the exercise performed. Estimated glucose uptake by the leg increased 7-fold after 40 min of light exercise and 10- to 20-fold at moderate to heavy exercise. Blood glucose uptake could at this time account for 28-37% of total substrate oxidation by leg muscle and 75-89% of the estimated carbohydrate oxidation.Splanchnic glucose production increased progressively during exercise reaching levels 3 to 5-fold above resting values at the heavy work loads. Close agreement was observed between estimates of total glucose turnover during exercise based on leg glucose uptake and splanchnic glucose production. Hepatic gluconeogenesis—estimated from splanchnic removal of lactate, pyruvate, glycerol, and glycogenic amino acids—could supply a maximum of 25% of the resting hepatic glucose production but could account for only 6-11% of splanchnic glucose production after 40 min of moderate to heavy exercise.It is concluded that: (a) blood glucose becomes an increasingly important substrate for muscle oxidation during prolonged exercise of this type: (b) peripheral glucose utilization increases in exercise despite a reduction in circulating insulin levels: (c) increased hepatic output of glucose, primarily by means of augmented glycogenolysis, contributes to blood glucose homeostasis in exercise and provides an important source of substrate for exercising muscle.