The transmural distribution of glucose uptake in the left ventricle of the heart in vivo was studied using the 2-deoxyglucose tracer method under conditions of resting and swimming. Intravenous tracer infusion and aortic blood sampling were performed through chronically implanted catheters. The ventricular glucose uptake of the resting rats averaged 1.5 mumol X min-1 X g protein-1 and was about 60% higher in the subendocardial layers than in the superficial layers (P less than 0.01). During a 20-min swimming period that increased the cardiac work load twofold, the glucose uptake rate was slower (P less than 0.001), about 1.0 mumol X min-1 X g protein-1, and evenly distributed across the left ventricular wall, whereas the reduction in glycogen stores, also evenly distributed through the ventricular wall, was 30 mumol glycosyl units/g protein. Blood lactate concentration increased from 1.8 to 10-13 mM and blood glucose from 6.1 to 12 mM during swimming, but plasma free fatty acid concentration decreased slightly. The inhibition of glucose uptake during swimming was probably caused by the increased use of other oxidizable substrates.