The mitochondrial membrane potential (Ψ_mito) is an important parameter not only of mitochondrial but also of cellular status. Prolonged mitochondrial depolarization is associated with various forms of neuronal death. Assessment of mitochondrial depolarization can take advantage of the specific properties of the lipophilic dyes that distribute in a potentiometrically determined ratio across membranes. Using conventional imaging, we showed that rhodamine 123 accumulated in the mitochondria, generating a highly heterogeneous pattern of spatial distribution of fluorescence across the cell body. Collapse of the Ψ_mito following exposure to a protonophore, carbonylcyanide p-chloromethoxyphenylhydrazone (CCCP), released rhodamine 123 from mitochondria into the cytosol. Under acutely changed conditions, this increased the overall intensity of the fluorescence signal and significantly decreased the degree of spatial heterogeneity of the signal. If mitochondrial depolarization was sustained chronically, the intensity of the signal decreased, but the increase in the spatial homogeneity of the fluorescent signal was maintained. Image analysis showed that the level of spatial heterogeneity of the signal can be assessed by calculating, for each individual neurone, the spread of pixel intensities values around the mean. This spread is defined by the coefficient of variation (CV), which is a measure of the standard deviation normalized to the average, and was inversely related to mitochondrial depolarization measured under different conditions. Thus, the degree of spatial heterogeneity of the rhodamine 123 signal measured from a neurone is a reliable indicator for the assessment of mitochondrial depolarization and can be used in experiments to monitorΨ_mito over shorter or longer periods.