We have previously reported the occurrence of pGlu-Glu-Pro-NH(2)(Glu-TRH, EEP), Val-TRH, Tyr-TRH, Leu-TRH, Phe-TRH, and Trp-TRH in rat brain using a combination of HPLC and radioimmunoassays with antibodies that cross-react with the general structure pGlu-X-Pro-NH(2) where 'X' maybe any amino acid residue (Peptides 2004; 25 : 647). This new family of TRH-like peptides, along with TRH (pGlu-His-Pro-NH(2)), has neuroprotective, anticonvulsant, antidepressant, euphoric, anti-amnesic, and analeptic effects. We now report that a combination of affinity chromatography using a rabbit antibody specific for Tyr-TRH and Phe-TRH, along with HPLC and tandem mass spectrometry operating in the multiple reaction monitoring (MRM) mode, provide conclusive evidence for the presence of Tyr-TRH in rat brain. Furthermore, synthetic Tyr-TRH is active in the Porsolt Swim Test suggesting that it is a fourth member of this family of in vivo neuroregulatory agents that have psychopharmacotherapeutic properties.