Active mitochondria were visualized with a laser scanning confocal microscope using the dye rhodamine 123, which is accumulated in mitochondria with a high respiratory activity. In glial precursor cells cultured from rabbit retina the fluorescence signal of active mitochondria was confined to the tips of processes, while in oligodendrocytes an even distribution was found. The developmental stage of cultured oligodendrocytes was determined by staining with O4 and O10 antibodies after the end of microfluorometric measurements. In glial precursor cells, the mitochondrial uncoupler CCCP generated an increase in the cytoplasmic calcium concentration ([Ca2+]i) in regions corresponding to those revealed by rhodamine 123 staining. In mature oligodendrocytes CCCP evoked [Ca2+]i elevation without significant spatial heterogeneity. However, electron microscopy did not reveal an uneven distribution of mitochondria in either immature or mature oligodendrocytes. We conclude that in the precursor cells, active mitochondria are confined to process tips and serve as a Ca2+ pool, while in oligodendrocytes such a spatial heterogeneity cannot be detected. This preferential localization of active mitochondria may play a role in the neurone-glia interaction during the formation of the contact between the myelinating glial process and the axonal membrane.