Retrograde labeling of neuronal elements in the brain and spinal cord has been investigated by autoradiographic techniques following injections of D-[3H]aspartate (asp), [3H] gamma-aminobutyric acid (GABA) or horseradish peroxidase (HRP) in the medulla and spinal cord of rats. Twenty-four hours after D-[3H]asp injections focused upon the cuneate nucleus, autoradiographic labeling is present over fibers in the pyramidal tract, internal capsule and over layer V pyramids in the forelimb representation of the sensorimotor cortex. After [3H]GABA injections in the same nucleus no labeling attributable to retrograde translocation can be detected in spinal segments, brain stem or cortex. Conversely, injections of 30% HRP in the cuneate nucleus label neurons in several brain stem nuclei, in spinal gray and in layer V of the sensorimotor cortex. These observations give further support to the proposed existence of a selective retrograde transport of D-[3H]asp and are consistent with the available evidence which indicates that the corticodorsal column nuclei path use glutamate and/or aspartate as neurotransmitter(s). D-[3H]Asp injections focused on the dorsal horn at cervical segments label a fraction of perikarya of the substantia gelatinosa and a sparser population of larger neurons in laminae IV to VI for a distance of 3-5 segments above and below the injection point. No brain stem neuronal perikarya appear labeled following spinal injections of D-[3H]asp although autoradiographic grains overlie pyramidal tract fibers on the side contralateral to the injection. This labeling however has not been observed rostral to lower pontine levels nor over cortical neurons at any of the survival times used in the present experiments (6-72 h). As in cases with cuneate injections this pattern of labeling contracts with that obtained after spinal injections of either [3H]GABA or HRP. Although labeling of neocortical neurons has not been observed after spinal injections of D [3H]asp, possibly as a result of the length of corticospinal axons, retrograde labeling of these elements for at least some distance may be taken as suggestive of a special affinity of their terminals for glutamate and/or aspartate.