Rat thyrotropin-releasing hormone prohormone (pro-TRH) contains five separate copies of the TRH progenitor sequence: Gln-His-Pro-Gly. Each of the five sequences is flanked by pairs of basic residues and linked together by one of several predicted connecting sequences. Two of the pro-TRH-connecting peptides, prepro-TRH-(160-169) and prepro-TRH-(178-199), were detected in extracts of rat neural tissues by radioimmunoassay using antibodies directed against the corresponding synthetic probes. Endogenous prepro-TRH-(160-169) and prepro-TRH-(178-199) were purified by gel exclusion chromatography, reverse-phase high pressure liquid chromatography, and ion-exchange chromatography. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards, immunological analysis, and tryptic mapping. Equimolar amounts of these connecting fragments were observed in hypothalamus and spinal cord. Quantification of TRH in spinal cord and hypothalamus extracts revealed the presence of 4.9-6.3 mol of TRH/mol of prepro-TRH-(178-199) and 4.4-6 mol of TRH/mol of prepro-TRH-(160-169), respectively. By using the indirect immunofluorescence technique, prepro-TRH-(178-199) immunoreactive cell bodies were found in the paraventricular nucleus of the hypothalamus, and a dense plexus of immunopositive nerve terminals was observed in the external zone of the median eminence, in a distribution similar to that described for TRH. These studies demonstrate that prepro-TRH-(160-169) and prepro-TRH-(178-199) are, together with TRH, predominant storage forms of the TRH precursor in hypothalamus and spinal cord, being present in molar ratios corresponding to those expected for a nearly complete processing of the prohormone molecule. The presence of pro-TRH-connecting peptides in various brain regions, including the median eminence, suggests that these peptides might act as neuromodulators in the central nervous system and/or neuroendocrine signals at the pituitary level. In the olfactory lobes, prepro-TRH is processed differently since a C-terminally extended form of TRH, prepro-TRH-(172-199), is found as a major end product along with lower but significant amounts of prepro-TRH-(178-199) and prepro-TRH-(160-169). The striking difference in pro-TRH processing patterns among the various tissues examined suggests differential regulating mechanisms for TRH and/or TRH-related activities.