Substance P, thyrotropin-releasing hormone (TRH) and serotonin are putative neurotransmitters which have been proposed to co-exist in some brain neurons. Our previous immunocytochemical and biochemical studies have demonstrated that 85–100% of all serotonin neurons are destroyed following neonatal 5,7-dihydroxtryptamine (5,7-DHT) treatment. In this study, we have determined the effect of neonatal 5,7-DHT and desipramine (DMI) treatment on the biochemical content and immunocytochemical localization of substance P and TRH throughout the brain. Interestingly, we have observed that virtually all substance P- and TRH-immunoreactive cells in the ventral pons-medulla are destroyed by the neurotoxin. However, peptide-containing neurons in other regions were not affected. Additionally, we measured the peptide content and found that TRH is significantly altered in any region, even after a pons-medulla (−20%), but not in other brain regions. Substance P content was not significantly altered in any region, even after a greater than 90% reduction of serotonin. These data indicate that the co-localized substance P and TRH forms a small porportion of the total peptide in brain.