Background. This study was designed to assess the protective effects of the mitochondrial adenosine triphosphate-sensitive potassium channel (K(ATP)) opener diazoxide as an additive to heart preservation solution. Methods. Forty isolated isovolumic buffer-perfused rat hearts were divided into four groups. Groups I and III hearts were arrested with and cold-stored in Celsior solution for 4 hr and 10 hr, respectively. In Groups II and IV, hearts underwent a protocol similar to that used in Group I and III, respectively, except that Celsior was supplemented with 100 μmol/L of diazoxide. Results. The protective effects of diazoxide were primarily manifest as a better preservation of diastolic function and a reduction of myocardial edema. The improvement of postischemic systolic function was observed only after prolonged exposure to diazoxide in Group IV, compared with Group IlI. The endothelium dependent and endothelium-independent coronary flow postischemic responses were not affected by the supplementation of Celsior with diazoxide. Conclusions. Pharmacologic activation of mitochondrial K(ATP) channels seems to be an effective means of improving preservation of cold-stored hearts, which is consistent with the presumed role of these channels as end effectors of the cardioprotective preconditioning pathway.