The distribution and the chemical identity of retrogradely single and double labeled neurons in the caudal raphe nuclei were analyzed in the rat following injection of two fluorescent tracers into the medial preoptic area and the ventral/intermediate grey of the lumbar spinal cord, and serotonin immunocytochemistry. The results suggest that (1) neurons in the caudal raphe nuclei exhibit highly collateralized axons, able to simultaneously innervate rostrally- and caudally-located targets; (2) a large proportion (40-50%) of the raphe-spinal projection does not contain serotonin, which by contrast is present in more than 70% of the neurons projecting to the medial preoptic area; (3) only a small fraction of the observed collateralized projection is serotonergic. Thus, multiple transmitter systems are likely to be involved in the diffuse ascending and descending influence arising from these nuclei.