Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to identify putative neurotransmitter(s) at the phrenic motor nucleus in the cat. Fast Blue was injected bilaterally into the diaphragm of five cats, where each phrenic nerve enters the muscle. Seven days later the animals were perfusion fixed and tissue sections from the fourth, fifth, and sixth cervical spinal cord segments were analyzed using a fluorescence microscope. Retrogradely labeled fluorescent phrenic motor neuron cell bodies appeared in all of the segments but primarily in sections from the fifth segment. The same or adjacent transverse sections were then used for the demonstration of the distribution of the neurotransmitters 5-hydroxytryptamine (5HT), substance P, and thyrotropin-releasing hormone (TRH) in the area of the phrenic motor nucleus using the indirect immunofluorescence technique. The most conspicuous neurotransmitters found at the phrenic motor nucleus were 5-HT and substance P. We observed dense and diffuse fiber networks throughout the ventral horn which contains the phrenic motor nucleus. These fibers contained varicosities in close proximity to phrenic motor neurons. In addition to 5-HT-and substance Pcontaining nerve endings, some fibers containing TRH were also found in the area of the phrenic motor nucleus. These results are consistent with earlier physiological data suggesting that 5-HT, substance P, and TRH are important neurotransmitters and/or neuromodulators involved in central control of respiration.Our knowledge of central respiratory neurons is extensive with regard to their anatomical localization and their electrophysiological properties (Cohen, 1979; Mitchell and Berger, 1981). However, very little is known about the neurotransmitters of these neurons. One approach commonly used to obtain information about this point has been to inject a putative CNS neurotransmitter, an agonist or antagonist of the neurotransmitter or a pre-