This study reports for the first time (a) the longitudinal profile of the transmembrane potential (mΔψ) of single mitochondria using a Nernstian fluorescent probe and (b) the distribution of mΔψ fluctuations of mitochondria undergoing permanent depolarization. Our findings show that (1) mitochondria in different energetic conditions coexist in the same cell, (2) mΔψ is rather homogeneous along the entire length of single mitochondria, (3) mΔψ is not influenced by the surrounding cytoplasmic environment and (4) mΔψ fluctuations occur simultaneously in groups of mitochondria connected in a network. Taken together, these findings provide further evidence for a functional relationship between mitochondrial arrangement and energetic condition.