Although there is considerable evidence that the analgesic action of electrical brain stimulation is mediated in part by serotonergic (5-HT) axons in the dorsolateral funiculus (DLF) of the spinal cord, studies in the rat have questioned the existence of this pathway. In this study, we used antisera directed against a conjugate of 5-HT and bovine serum albumin (BSA) to identify immunoreactive 5-HT axons in the DLF of the rat and cat. Both light and electron-microscopic studies were performed so that the fiber caliber of the labeled axons could also be determined. We found a rich complement of immunoreactive 5-HT axons in the DLF of both rat and cat. Although these could be seen without difficulty in the normal cat, in the rat it was necessary to make a lesion of the DLF to build up the staining rostrally. Ultrastructural analysis established that almost all of the labeled axons (in rat and cat) were unmyelinated. We conclude that there are indeed 5-HT immunoreactive axons in the DLF of the rat and cat. These presumably derive from neurons of the medullary nucleus raphe magnus (NRM), which have been implicated in the descending controls exerted by opiates and electrical brain stimulation. The results suggest that previous physiological studies of the properties of the opiate-responsive, spinally projecting NRM neurons were not made from those that are 5-HT containing.