A rapid and sensitive chromatographic method is presented for determining TRH and TRH-like peptides in small quantities of tissues and fluids. The procedure was applied to screen a comprehensive range of tissues from the central nervous system (CNS) and periphery of the rat. When hydrochloric acid (100 mmol/l) was used for extraction, the TRH immunoreactivity obtained was identified solely with the known tripeptide hormone. In contrast, when weakly acidic conditions were used for extraction, neutral or acidic TRH-like peptides which lacked the histidine residue at position 2 of the hormone, but reacted with TRH antibodies, were shown to be present in addition to TRH. The TRH-like peptides in the brain were located principally in the hippocampus, brain stem and dorsal colliculi. In the periphery, TRH-like peptides were shown to be present in the male reproductive system and certain endocrine tissues. In addition, the presence of TRH-like peptides was confirmed in portal blood. The results indicate that peptides with a neutral or acidic TRH-like sequence at their C-terminus are widely distributed in the CNS and periphery of the rat. These peptides appear to occur mainly or exclusively in precursor forms from which TRH immunoreactivity can be released during extraction under weakly acidic conditions.