Thyrotrophin-releasing hormone (TRH), a hypothalamic neuropeptide hormone and a putative neuromodulator/neurotransmitter in the central nervous system is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a TRH-specific metallopeptidase localized on the surface of neuronal brain cells in culture and on lactotrophic cells of the pituitary. After succeeding in cloning the cDNA of TRH-DE we now report on the cellular distribution pattern of this enzyme in rat brain, spinal cord and pituitary gland using in situ hybridization histochemistry. In the pituitary, TRH-DE mRNA was found both in the anterior and the neural lobe but not in the intermediate lobe. After treatment with triiodothyronine (T3) a dramatic increase in the mRNA levels of the TRH-DE and a decrease in the intensity of the TRH receptor could be observed in the anterior lobe of the pituitary. In brain, TRH-DE transcripts were predominantly found in neo- and allocortical regions with strongest signals in the olfactory bulb, the piriform cortex, the cerebral cortex, the granular layer of the cerebellar cortex and the pyramidal cells of the Ammon's horn. In the diencephalon, the highest TRH-DE mRNA levels were observed in the medial habenulae followed by several hypothalamic subregions. In the mesencephalon and brainstem, moderate signals were present in the superior colliculi, substantia nigra, dorsal raphe and in the periolivar region. In the spinal cord, TRH-DE mRNA positive neurons were present in all layers. The very distinct distribution of TRH-DE in the brain and the hormonal regulation of the adenohypophyseal enzyme support the concept that this peptidase serves very specialized functions.