This study examines the electrophysiological and metabolic changes that occur in rabbit hearts during hypothermic storage in vitro . Hearts were microperfused at 4°C for 6 or 24h with either normal Krebs-Henseleit buffer (KHB) or KHB containing 2,3-butanedione monoxime (BDM). After hypothermic storage, hearts were rewarmed to 37°C with KHB. Cardiac function was then assessed in Langendorff perfusion mode. Electrophysiological changes were also assessed from the ventricular paced-evoked responses. After storage, mitochondria were isolated from the hearts and their respiratory control ratio, rate of ATP synthesis and outer membrane intactness were assessed. Compared with values from fresh non-stored hearts, hearts stored hypothermically for 24h showed significant decreases in both left ventricular developed pressure and coronary flow when reperfused in Langendorff mode. On the other hand, the decrease in left ventricular developed pressure in hearts that were stored for only 6h (with or without BDM) was not significant. Compared with values obtained from fresh non-stored hearts, hypothermic storage significantly decreased the R-wave amplitude, and both the R-E and ST-E intervals of paced-evoked responses. This was true for hearts microperfused for 6h (with or without BDM) and for hearts microperfused with buffer containing BDM for 24h. The ST-R intervals in hearts microperfused hypothermically for 6h were prolonged, but this change was not statistically significant compared with those obtained from unstored hearts. In hearts microperfused with KHB containing BDM for 24h, the ST-R interval was significantly prolonged. Hypothermic microperfusion for 24h significantly decreased both the mitochondrial coupling ratio and the rate of ATP synthesis. In hearts microperfused with BDM for 6h, mitochondrial coupling ratios and the rate of ATP synthesis were not significantly different from those in fresh hearts. In conclusion, the present study has shown that long-term hypothermic storage significantly impaired both paced-evoked responses and mitochondrial function. Inclusion of BDM in the perfusion buffer during storage significantly ameliorated some of these changes.