The localization of serotonergic, various peptidergic and possibly cholinergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemical and histochemical techniques. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releasing hormone-like immunoreactivity and for the histochemical marker acetylcholinesterase were all observed in each of the raphe nuclei of the medulla, as well as in the adjacent ventrolateral reticular formation. The similar distributions of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe-spinal neurons contain serotonin co-existing with one or more peptides in the same cell.