Previous studies have demonstrated that when tritiated proline [( 3H]Pro) is injected into the dorsal column nuclei (DCN) of cats, it labels macroglial cells, but fails to label neurons at the injection site. (Tritiated leucine [( 3H]Leu) in contrast, labels both neurons and some glial cells.) Despite the failure of [3H]Pro to label DCN neurons, labeling is still observed in DCN terminal targets. This result suggests that glial cells are involved in the translocation of [3H]Pro-labeled molecules from one part of the brain to another. The purpose of the present experiment was to use electron microscopic autoradiographic techniques to characterize the labeling produced in internal arcuate fiber tract axons arising from DCN neurons 24 h after injections of [3H]Pro (or [3H]Leu, for comparison) into DCN. It was reasoned that, if the translocation of [3H]Pro-labeled molecules from DCN to its targets is indeed carried out by glial cells, then only glial elements associated with the fibers should be labeled following [3H]Pro injections of DCN. If, on the other hand, the translocation involves an initial transfer of [3H]Pro-labeled molecules into neuronal perikarya followed by axonal transport, then only axoplasmic elements along the fiber pathway should be labeled. Injections of [3H]Pro into DCN labeled axoplasmic elements in samples of axons from the internal arcuate tract both 'near' (0.5-0.8 mm) and 'far' (2-4 mm) from the injection site at about an equal absolute density. However, glial elements associated with the axons were also labeled in both samples, but much more densely in the 'near' than in the 'far' axons. Injections of [3H]Leu labeled axoplasm more densely than did [3H]Pro (by a factor of 4 in the 'far' samples). Glial labeling by [3H]Leu near the injection site was much less than that of [3H]Pro, but, 'far' from the injection, the levels of [3H]Leu and [3H]Pro glial labeling were comparable. Taken together with the results of other studies, these data support the existence of a previously unrecognized system of communication between glial cells and neurons. In this putative system (Fig. 9), molecules containing both [3H]Leu and [3H]Pro are transferred from glial cells into adjacent neuronal soma and transported down the length of the axon where, all along the way, some of them are transferred from the axon into adjacent glial processes. The system is more readily apparent when [3H]Pro is used because of its avid and preferential uptake by glial cells. Potential functions of such a system are unknown, but could be trophic, protective and/or informative.