Background Pinacidil solutions have been shown to have significant cardioprotective effects. Pinacidil activates both sarcolemmal and mitochondrial potassium-adenosine triphosphate (K ATP) channels. This study was undertaken to compare pinacidil solution with University of Wisconsin (UW) solution and to determine if the protective effect of pinacidil involved mitochondrial or sarcolemmal K ATP channels. Methods Thirty-two rabbit hearts received one of four preservation solutions in a Langendorff apparatus: (1) UW; (2) a solution containing 0.5 mmol/L pinacidil; (3) pinacidil with Hoechst-Marion-Roussel 1098 (HMR-1098), a sarcolemmal channel blocker; and (4) pinacidil with 5-hydroxydecanote, a mitochondrial channel blocker. Left ventricular pressure-volume curves were generated by an intraventricular balloon. All hearts were placed in cold storage for 8 hours, followed by 60 minutes of reperfusion. Results Postischemic developed pressure was better preserved by pinacidil than by UW. This cardioprotective effect was eliminated by 5-hydroxydecanote and diminished by HMR-1098. Diastolic compliance was better preserved by pinacidil when compared with UW. This protection was abolished by the addition of 5-hydroxydecanote and moderately decreased by HMR-1098. Conclusions Our results support the superiority of pinacidil over UW after 8 hours of storage. The cardioprotective role of pinacidil is mediated primarily by the mitochondrial K ATP channel.