Objective: The aim was to investigate the effects of chronic right ventricular pressure overload on myocardial glucose and free fatty acid metabolism in the right ventricular free wall, ventricular septum, and left ventricular free wall. Methods: Using a glucose analogue, ¹⁴C-2-deoxyglucose (¹⁴C-DG), and a fatty acid analogue, ¹⁴C-β methylheptadecanoic acid (¹⁴C-BMHDA), quantitative autoradiography was performed in conscious rats with 4 week pulmonary artery constriction. Results: In rats with chronic pulmonary artery constriction, right ventricular peak systolic pressure and right ventricular weight to body weight ratio increased by 88% and 127%, respectively, compared with sham operated rats (P < 0.01 for each). In the right ventricular free wall, ¹⁴C-DG deposition increased but ¹⁴C-BMHDA accumulation did not differ in the chronic pulmonary artery constricted rats compared with sham operated rats [212(SEM 27), n = 6 ν 101(15) nCi·g₋₁, n = 4, P < 0.01, and 406(40), n = 6, ν 333(48) nCi·g₋₁, n = 4, NS, respectively]. In sham operated rats, ¹⁴C-DG and ¹⁴C-BMHDA deposition did not differ between the ventricular septum and the left ventricular free wall. In contrast, ¹⁴C-DG and ¹⁴C-BMHDA accumulations were lower in the ventricular septum compared with the left ventricular free wall wall in chronic pulmonary artery constricted rats. Myocardial blood flow assessed by ¹⁴C-iodoantipyrine was homogeneously distributed throughout both ventricles. Conclusions: Chronic right ventricular pressure overload increases myocardial glucose uptake and/or its phosphorylation in the right ventricular free wall, and alters the regional profiles of substrate use in the ventricular septum and left ventricular free wall despite the homogeneous blood flow distribution. The results of the acute right ventricular pressure overload study, in which only right ventricular ¹⁴C-BMHDA deposition was increased compared with controls, suggest that the findings obtained from chronic pulmonary artery constricted rats cannot be explained by increased right ventricular pressure alone.