The effects of lactate and free fatty acids (oleate) on the transmural distribution of glucose uptake in the left ventricular wall of the isolated perfused rat heart were studied using the deoxyglucose tracer method. The left ventricular glucose uptake was 2.9 +/- 0.3 mumol/min per gram protein (mean +/- S.D.) in the control heart and about 30% higher in the innermost third of the ventricular wall than in the more superficial layers (P less than 0.001). The addition of lactate (5 or 15 mM) or oleate (0.7 mM) to the perfusate decreased the mean myocardial glucose uptake by 40% to 50% (P less than 0.01), but the subendocardial: subepicardial glucose uptake ratio remained unaltered. Elevation of the aortic pressure from 7.85 kPa to 14.7 kPa resulted in an increase of 65% (P less than 0.01) in the average cardiac glucose uptake and caused the transmural uptake gradient to disappear. The present results indicate that although total cardiac glucose utilization is influenced by both the mechanical work load and the substrate supply, it is the former that determines the transmural distribution of glucose uptake.