The relationship of serotonergic neurons in raphe and ventrolateral medullary regions to neurons projecting to the rostral ventral respiratory group in the rat were investigated using combined immunohistochemical and retrograde labeling techniques. Serotonergic and non-serotonergic neurons retrogradely labeled with rhodamine beads were found intermingled in the larger population of serotonin-immunoreactive neurons in raphe pallidus, obscurus and magnus. In addition, a cell group analogous to the retrotrapezoid nucleus in the cat was identified in the rat ventrolateral medulla. Retrotrapezoid neurons, which exhibited exclusively ipsilateral projections to the ventral respiratory group, were located lateral to clusters of serotonergic cells near the ventral surface, but were not serotonin immunoreactive.