Immunohistochemical reactions for 12 putative neuromessengers combined with retrograde labeling of phrenic motoneurons identified seven neuromessengers (5-hydroxytryptamine, substance P, thyrotropin-releasing hormone, methionine enkephalin, cholecystokinin, galanin, neuropeptide Y) located within terminal varicosities in the phrenic nucleus. The degree of terminal labeling in the phrenic nucleus varied depending on the peptide. Substance P, thyrotropin-releasing hormone and methionine enkephalin were each tested for colocalization with 5-hydroxytryptamine within terminal varicosities in the phrenic nucleus, and the coincidence of double-labeling varied for each peptide. These results indicate that phrenic motoneurons are subject to modulation by many peptide neuromessengers that may alter their responsiveness to primary excitatory and inhibitory inputs.