Thyrotropin-Releasing Hormone (TRH) is an important extracellular signal substance that acts as a stimulator of hormone secretion from adenohypophyseal target cells and fulfills many criteria for the function of a neuromodulator/neurotransmitter within the central and peripheral nervous systems. The inactivation of TRH-signals is catalysed by a highly specific ectoenzyme. Here, we characterize the human TRH-degrading ectoenzyme (TRH-DE) by primary sequence, functional expression, genomic structure and chromosomal assignment. By screening a cDNA-library constructed from human lung, 5.7 kb of cDNA were identified. The longest open reading frame predicts a type II integral membrane protein of 117 kDa. The extracellular domain contains the HEXXH + E motif that is characteristic of a certain family of Zn-dependent aminopeptidases. Within this family, the sequences of human and rat TRH-DE reveal an unusual high degree of conservation (96% identical residues). Specific enzymatic activity was observed after transfecting COS-7 cells with human TRH-DE cDNA yielding a Km for TRH hydrolysis of 29.7 microM. Northern blot analysis demonstrated a restricted tissue distribution with highest transcript levels in the brain. Using fluorescent in situ hybridization with the cDNA and a genomic lambda clone, respectively, we localized the TRH-DE gene to the long arm of human chromosome 12. Five independent P1 artificial chromosome clones were required to span the complete cDNA sequence and revealed that it is distributed on 19 exons. Interspecies Southern analysis suggests that the gene is present as a single copy in human, monkey, rat, mouse, dog, bovine, rabbit and chicken DNA. All of these data further the notion that the TRH-DE is not an ordinary enzyme but a specific neuropeptidase that has been highly conserved among species.