The present study investigated the ventral medullary distribution of enkephalin-immunoreactive neurons that project to the intermediolateral cell column and the relationship of these neurons to substance P-immunoreactive neurons. Neurons that projected to the intermediolateral cell column were identified by the presence of rhodamine-labeled microspheres within the neuronal cell body after an injection of the microspheres into the intermediolateral cell column of the third thoracic spinal cord segment. Enkephalin- and substance P-immunoreactivities were identified by dual-color immunohisto-chemistry. Enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were present in the raphe magnus, the nucleus reticularis magnocellularis pars alpha, the paragigantocellular reticular nucleus, and the parapyramidal region. These neurons were present throughout the rostrocaudal extent of each of these nuclei. However, in the raphe magnus the greatest number was present at more rostral levels of the nucleus. The morphology and distribution of enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were similar to those of enkephalin-immunoreactive neurons that were not observed to contain rhodamine-labeled microspheres. Substance P- and enkephalin-immunoreactive neurons that projected to the intermediolateral cell column were present in similar distributions in each of the nuclei studied, except the raphe magnus. The raphe magnus contained more enkephalin- than substance P-immunoreactive neurons at rostral levels and more substance P-immunoreactive neurons than enkephalin-immunoreactive neurons at caudal levels. Coexistence of substance P- and enkephalin-immunoreactivities in ventral medullary neurons that projected to the intermediolateral cell column was rarely seen. These studies support the hypothesis that ventral medullary enkephalinergic neurons project to the intermediolateral cell column where they could act to modulate preganglionic sympathetic activity.