Experiments were performed on 6 adult cats. Brainstem neurons that project to the phrenic nucleus were retrogradely labeled by microinjection of WGA-HRP into the spinal phrenic nucleus. The brainstem sections were double processed for HRP histochemical staining with TMB-DAB method and 5-HT immunohistochemical staining with anti-5-HT primary antibody and FITC conjugated immunofluorescent secondary antibody. HRP-5-HT double-labeled neurons were observed mainly in the nucleus raphe pallidus. A few double-labeled neurons were also observed in nucleus raphe obscurus and nucleus raphe magnus. 5-HT immunopositive terminal fibers were observed in the vicinity of HRP single-labeled neurons (phrenic premotor neurons) located in the nucleus ambiguus and ventrolateral solitary tract nucleus. The results indicate that the 5-HTergic neurons in the nucleus raphe pallidus send axonal projections to the phrenic nucleus, and that the brainstem phrenic premotor neurons also receive 5-HTergic axonal projections.