By use of the indirect immunofluorescence technique the distributions of 5-hydroxytryptamine (5-HT)-, substance P- and thyrotropin-releasing hormone (TRH)-immunoreactive (IR) neurons have been studied in the midline raphe nuclei and nucleus reticularis lateralis of the caudal brainstem (levels P18.3-P8.5; according to Berman (1968), in the cat, after treatment with colchicine. In addition, by use of the double-labelling technique, the coexistence between 5-HT-, substance P- and TRH-like immunoreactivity (LI) in these neurons was analysed. The results show that cell bodies in the midline raphe nuclei and nucleus reticularis lateralis contain 5-HT-, substance P- and TRH-LI. 5-HT-IR cells were more abundant than peptidergic neurons in all areas analysed. Quantitative estimations indicated that the total number of 5-HT-IR cells in the regions studied was about 17 x 10(3), while the corresponding numbers for substance P- and TRH-IR cells were 11 x 10(3) and 12 x 10(3), respectively. From double-labelled sections it was concluded that the vast majority of peptidergic cells also contained 5-HT-LI (87-100%). However, a subpopulation of 5-HT-IR neurons lacked peptide-LI (10-55%). The degree of coexistence varied with the brainstem level, in that neurons at more rostral locations showed a lower incidence of coexistence between 5-HT and peptide(s). The presence of all three compounds in one and the same cell body could also be demonstrated. In summary, 5-HT-, substance P- and TRH-IR cell bodies were encountered in medullary nuclei known to contain neurons with projection to the spinal cord. A high degree of coexistence between the compounds was demonstrated in these nuclei. The obtained results fit well with earlier studies on the patterns of distribution and peptide colocalization of 5-HT fibres in the spinal cord. The existence of biochemically distinct neuronal subpopulations within the 5-HT bulbospinal pathway is discussed.