The origins of the serotonergic projections to the spinal cord in the rat were determined by employing the retrograde cell marker HRP coupled with the unlabeled antibody, peroxidase-antiperoxidase immunocytochemical method of Sternberger. Large numbers of stained neurons (greater than 70%) in the medullary raphe nuclear complex were found to contain both HRP retrogradely transported from the spinal cord and positive 5-HT staining. These serotonergic cell groups, including the nucleus raphe obscurus, raphe pallidus, raphe magnus, and the ventral parts of the reticular formation, project to all spinal cord levels. In addition, some neurons contained HRP granules, but were unstained for 5-HT, suggesting that they may contain other non-serotonergic neurotransmitters. More rostrally in the midbrain reticular formation, many 5-HT neurons were found to have projections exclusively to the cervical spinal cord. These findings indicate that the descending serotonin inputs to the spinal cord originate not only from the serotonergic neurons located in the medullary raphe complex, but also from other new sources located in the central gray and reticular formation of the midbrain.