A cDNA encoding prepro-thyrotropin-relaesing hormone (ppTRH) in chicken (Gallus gallus) was isolated and the sites of expression in the brain were determined. The chicken ppTRH cDNA encodes 260 amino acids, including four TRH progenitor sequences (-Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg-). It is interesting to note that chicken ppTRH harbors four TRH progenitor-like sequences. According to the hydropathy profile of chicken ppTRH, not only the TRH progenitor sequences but also the TRH progenitor-like sequences are localized in hydrophilic regions. The TRH progenitor-like sequences might be related to structural conservation in the evolution of ppTRH, although they cannot be processed into TRH due to the mutation of several amino acids. According to the alignment of the deduced amino-acid sequences of known vertebrate ppTRHs and the molecular phylogenetic tree we constructed, we speculate on the molecular evolution of ppTRH in vertebrates. In situ hybridization demonstrated experession of the ppTRH gene in the nucleus preopticus periventricularis, nucleus preopticus medialis, regio lateralis hypothalami, paraventricular nucleus, nucleus periventricularis hypothalami, and nucleus ventromedialis hypothalami in the chicken brain.