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Pyroglutamyl-N^3im-methyl-histidyl-prolineamide (methyl-thyrotrophin releasing hormone, methyl-TRH) is a potent synthetic analogue of TRH. N^3im-Methyl-histidine is present in mammalian brain and it has been suggested that methyl-TRH is a physiological releasing hormone normally present in the hypothalamus. A non-gradient cation-exchange chromatography system that uses SP-Sephadex C-25 and completely resolves methyl-TRH and TRH has been developed. Because methyl-TRH cross-reacts in the immunoassay for TRH, this assay was used to measure TRH and methyl-TRH in the chromatographic fractions. By this means it has been demonstrated that the amount of methyl-TRH present in the rat is less than 0·025 ng/hypothalamus.