Following injection of horseradish peroxidase-labeled wheat germ agglutinin or of rhodamine-labeled microspheres as non-selective tracers into the rat red nucleus, the origins of the corticorubral and cerebellorubral pathways, as well as a considerable number of other brain structures including dorsal raphé nucleus, zona incerta and several hypothalamic nuclei showed retrogradely labeled perikarya. Labeling patterns obtained with horseradish peroxidase-labeled wheat germ agglutinin compared well with those observed following application of rhodamine-labeled microspheres which produced injection sites restricted to the small nucleus. In these latter cases, counterstaining with phosphine allowed a better definition of anatomical structures. After D-[3H]aspartate application, retrogradely labeled perikarya were observed in cerebral cortex (layer V), zona incerta, dorsal raphé nucleus and in several other structures also labeled by non-selective tracers. Following application of [3H]choline and using an improved autoradiographic method, perikaryal labeling was massive within nucleus interpositus, while it was absent in dorsal raphé nucleus, cerebral cortex and zona incerta. Retrograde tracing experiments with D-[3H]aspartate and [3H]choline revealed that these transmitter related compounds are selective markers for two subsets of afferents to the red nucleus. The transmitter specificity of the selective labeling with [3H]choline in the cerebellorubral pathway is supported only in part by the results obtained with other methods. The selective labeling with D-[3H]aspartate in the corticorubral pathway, on the other hand, is consistent with its transmitter specificity.