It is now clear that TRH is derived from posttranslational processing of a precursor polyprotein like other hypothalamic releasing factors and not by a soluble nonribosomal enzymatic mechanism. With an oligonucleotide probe directed against a presumptive TRH progenitor sequence, a frog skin cDNA library was screened and a clone identified coding for a peptide of 123 amino acids containing four copies of the TRH progenitor sequence (Gln-His-Pro-Gly) flanked by paired basic amino acid residues. The amphibian probe did not, however, hybridize with mammalian hypothalamus. To identify the TRH precursor in the rat hypothalamus, an antiserum was raised against the synthetic decapeptide sequence, Cys-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Cys. It was hypothesized that the N- and C-terminal cysteines would cyclize, permitting an antibody to be generated against the midregion of the molecule and its extended counterpart sequences in nature pro-TRH. Such an antiserum was generated that recognized the intact or partially processed precursor immunohistochemically and was used to identify the prepro-TRH cDNA on screening of a rat hypothalamic lambda gt11 expression library. The rat precursor is similar to the amphibian only insofar as multiple copies of the TRH sequence are encoded in each. Thus, the resolution of the contentious question of the mode of TRH biosynthesis in the rat hypothalamus required the development of a novel antiserum, screening by immunocytochemistry and the application of modern molecular biological techniques.