A radioimmunoassay for thyrotropin-releasing hormone (TRH) precursor peptide Lys-Arg-Gln-His-Pro-Gly-Arg-Arg (pro-TRH), has been developed. Anti-pro-TRH antibody was raised in rabbits immunized with a conjugate of synthetic pro-TRH analog, Cys-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Cys (pCC10) to bovine serum albumin. This antibody did not cross-react with TRH, TRH-OH, His-Pro-diketopiperazine, neuropeptides, pituitary hormones and peptides which are included in prepro-TRH. Radioiodination of pCC 10 was performed by chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Pro-TRH was extracted from tissues, using 1.0 N acetic acid. The assay was performed with a double antibody system. The values are expressed as an equivalent of pCC 10. The dilution curves of acetic acid-extracts of rat hypothalamus and stomach in radioimmunoassay system were parallel to the standard curve. The recovery of tissue pro-TRH was 80%, the intra-assay and interassay variation was 5.2% and 8.9%, respectively. The elution profiles of acetic acid-extracts of the rat hypothalamus and stomach on Sephadex G-50 showed a single peak corresponding that of pCC 10. Immunoreactive pro-TRH was found in the rat brain, spinal cord, eye, stomach, intestine, pancreas and adrenal gland. These data suggest that this assay system is a suitable to measure pro-TRH in the tissues, and that pro-TRH is widely distributed in the rats.