TRH occurs in very high concentration in rat prostate. A species specific protein with repetitive -Gln-His-Pro-Gly- sequences, which are flanked on the N- and C-terminus by paired basic residues, has been shown to be the source of TRH in frog skin and rat hypothalamus. Following cleavage by trypsin-like enzymes, the peptide fragments with N-terminal Gln spontaneously cyclize to pGlu while Gly within the C-terminally extended peptides serves as the -NH2 donor for the alpha-amidation of the proline residue. Because this last step in the biosynthesis of TRH is rate limiting for pGlu-His-Pro-Gly, we have combined several chromatographic and radioimmunoassay techniques to identify this TRH precursor in rat prostate.