BACKGROUND: Mitochondria play a critical role in ischemia-reperfusion injury of the heart. The purpose of the present study was to analyze the intracellular region-specific functional state of mitochondria after cold ischemia-reperfusion in a rat heart transplant model. METHODS: Imaging of the mitochondrial functional state in situ in nonfixed myocardial fibers was performed by confocal microscopy of mitochondrial flavoprotein autofluorescence as redox state indicator; fluorescence of Rhod-2, a specific probe for mitochondrial calcium; and of tetramethylrhodamine ethyl ester fluorescence to monitor the mitochondrial membrane potential. RESULTS: This imaging demonstrated that, in contrast to control fibers, 10-hr heart cold storage, heterotopic cardiac transplantation, and 24-hr reperfusion result in a highly heterogeneous mitochondrial functional state (mitochondrial calcium content, redox state, and inner membrane potential), thus suggesting local permeability transitions and heterogeneous mitochondrial damage. CONCLUSIONS: Imaging of in situ mitochondria allows topologic assessment of mitochondrial defects and heterogeneity, consequently providing new insights into the mechanisms of cardiac ischemia-reperfusion injury.