Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a pivotal role in early and late ischemic preconditioning, but the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in rat hearts using confocal microscopy, immunofluorescence, and Western blot analysis. The green fluorescent probe glibenclamide-BODIPY was colocalized with the red fluorescent mitochondrial marker MitroTracker Red in isolated ventricular myocytes and in ventricular myocyte mitochondria, indicating the presence of sulfonylurea receptors (SURs) in the mitochondria. Anti-Kir6.1, anti-Kir6.2, and anti-SUR2 immunofluorescence was colocalized with that of MitoTracker Red in isolated mitochondria, suggesting that Kir6.1, Kir6.2, and SUR2 subunits are present in the mitochondria. Similarly, Kir6.1 (approximately 46 kDa), Kir6.2 (approximately 46 and approximately 40 kDa), and SUR2 (approximately 140 kDa) proteins were found to be expressed in mitochondria using Western blot analysis. By contrast, SUR1 was not present in mitochondria. These results suggest that mitoKATP channels in rat hearts might comprise a combination of Kir6.1, Kir6.2, and SUR2 subunits.