Isolated rat liver mitochondria were incubated under various metabolic conditions to determine their membrane potential (MMP) as measured continuously by a tetraphenylphosphonium (TPP+)-selective electrode. By flow cytometry, a parallel analysis of fluorescence emissions observing single mitochondria stained with the lipophilic cation 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) revealed linear correlation between the median orange fluorescence (FL2) due to J-aggregate formations and MMP values measured by TPP+. No correlation was detected with the green fluorescence (FL1) emission. A significantly higher correlation appeared between the FL2/FL1 ratio and MMP values. Within the same mitochondrial population, cytofluorimetric analysis revealed the presence of various classes of organelles with different MMP, whose distribution was dependent on metabolic condition. The highest functional heterogeneity was found in deenergized mitochondria, while the highest homogeneity was observed during the first phase of the phosphorylative process. Thus, these data suggest that the cytofluorimetric use of JC-1 provides direct experimental evidence for the hypothesis of functional mitochondrial heterogeneity, at least with respect to their membrane potential.