Meaning interpretation of metabolic images obtained by positron emission tomography for evaluation of cardiac disease requires a knowledge of the normal variation in regional myocardial substrate metabolism. Recent studies with fluorine-18 (F-18) fluorodeoxyglucose suggest inhomogeneity of myocardial glucose metabolism in the normal human heart, which may relate to substrate availability. Therefore, quantitative evaluation of myocardial oxidative metabolism and glucose metabolism, as derived by dynamic positron emission tomography with carbon-11 (C-11) acetate and F-18 fluorodeoxyglucose, was performed in nine healthy male volunteers. All were studied under tightly controlled metabolic conditions of hyperinsulinemic-euglycemic clamping with and without a concurrent lipid emulsion infusion. Significant inhomogeneity of regional glucose metabolism was noted although it was less than that described under fasting conditions. Glucose utilization was 13% lower in the septum compared with the lateral wall both without and with lipid infusion (0.34 vs. 0.39 mumol/g per min, respectively, p less than 0.05; and 0.33 vs. 0.38 mumol/g per min, respectively, (p less than 0.05). Relatively decreased septal glucose utilization could not be explained by decreased metabolic demand because C-11 clearance constants were marginally higher in the septum than in the lateral wall in both studies (0.055 vs. 0.054 per min, respectively, p = NS; and 0.061 vs. 0.056 per min, respectively, p less than 0.05). Relatively decreased septal glucose utilization could reflect regional variation in substrate use and possible preferential free fatty acid utilization by the septum. These data provide a useful framework for assessing altered cardiac metabolism in disease and support standardization of metabolic conditions during metabolic imaging with positron emission tomography.