The spinal projections from the raphe-associated brainstem areas containing serotonergic neurons were studied with aldehyde-induced fluorescence in combination with the retrograde fluorescent tracer True Blue in the rat. This technique makes it possible to determine simultaneously the projections of individual neurons and to detect whether serotonin is present in the same neurons. After tracer injections into the spinal cord retrogradely labeled serotonergic and non-serotonergic neurons were found in the medullary raphe nuclei and adjacent regions and to a lesser extent in association with the dorsal and median raphe nuclei in the mesencephalon. Large True Blue injections that covered one side of the spinal cord at mid-cervical level labeled about 60% of the ipsilaterally situated serotonergic neurons in the medullary raphe regions while the corresponding figure contralaterally was about 25%. On both sides a larger number of labeled non-serotonergic neurons were found; these were sometimes located dorsal to, but often intermingled with, the serotonergic cells. While the serotonergic projection from the mesencephalon could not be labeled from injections below cervical levels, the labeling in more caudal brainstem regions exhibited only minor variations depending on the rostrocaudal level of the spinal segment injected. Furthermore, quantitative data from injections at different levels indicate that the majority of the spinal-projecting neurons traverse most of the length of the cord. Summarizing the results obtained from small injections restricted to subregions of the cord we feel that it is possible to distinguish three fairly distinct pathways for spinal projections from the medullary raphe and adjacent regions: The dorsal pathway originates mainly from cells in the caudal pons and rostral medulla oblongata (rostral part of nucleus raphe magnus, nucleus raphe magnus proper, nucleus reticularis gigantocellularis pars alpha and nucleus paragigantocellularis). This pathway, which contains a large non-serotonergic component, descends through the dorsal part of the lateral funiculus and terminates mainly in the dorsal horn at all spinal cord levels. The intermediate pathway is largely serotonergic with its cell bodies located within the arcuate cell group (situated just ventral and lateral to the pyramids very close to the ventral surface of the brainstem) and in the nucleus raphe obscurus and pallidus and terminates in the intermediate grey at thoracolumbar and upper sacral levels.(ABSTRACT TRUNCATED AT 400 WORDS)