Oligodendrocytes can myelinate a variable number of axons in their surroundings; however, the mechanisms underlying axon-oligodendrocyte associations are unknown. We tested the hypothesis that single oligodendrocytes exclusively myelinate axons belonging to the same functional system. Carbocyanine dyes (DiI, FAST DiI) were applied to the sternomastoid muscle of the rat and allowed to transport retrogradely for 4 weeks within the motor axons. Using fluorescence microscopy and iontophoretic injection of Lucifer Yellow (LY), oligodendrocytes were injected in the proximity of retrogradely carbocyanine dye-labeled axons. Using dual channel confocal laser scanning microscopy (CLSM), the three-dimensional relationship between axons and glia was studied. The data indicate that a single oligodendrocyte can myelinate retrogradely labeled axons concomitantly with other, unlabeled axons belonging to apparently different functional systems.