Diabetic cardiomyopathy is the leading cause of heart failure among diabetic patients, and mitochondrial dysfunction has been implicated as an underlying cause in the pathogenesis. Cardiac mitochondria consist of two spatially, functionally, and morphologically distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). SSM are situated beneath the plasma membrane, while IFM are embedded between the myofibrils. The goal of this study was to determine whether spatially distinct cardiac mitochondrial subpopulations respond differently to a diabetic phenotype. Swiss Webster mice were subjected to intraperitoneal injection of streptozotocin or citrate saline vehicle. Five weeks following injection, diabetic hearts displayed decreased rates of contraction, relaxation, and left ventricular developed pressures (P<0.05, for all three). Mitochondrial size (FSC; P<0.01) and complexity (SSC; P<0.01) were both decreased in diabetic IFM, but not in diabetic SSM. Electron transport chain complex II respiration was decreased in diabetic SSM (P<0.05) and diabetic IFM (P<0.01), with the decrease being greater in IFM. Further, IFM complex I respiration and complex III activity were decreased with diabetes (P<0.01), but were unchanged in SSM. Superoxide (O₂•-) production was increased only in diabetic IFM (P<0.01). Oxidative damage to proteins and lipids, indexed through nitrotyrosine residues and lipid peroxidation, were higher in diabetic IFM (P<0.05 and P<0.01, respectively). The mitochondrial-specific phospholipid, cardiolipin, was decreased in diabetic IFM (P<0.01), but not in SSM. These results indicate that diabetes mellitus imposes a greater stress on the IFM subpopulation which is associated, in part, with increased O₂•- generation and oxidative damage, resulting in morphological and functional abnormalities that may contribute to the pathogenesis of diabetic cardiomyopathy.