Using a combination of PCR and hybridization screening, we have isolated a cDNA clone for a metalloendopeptidase (h-MP78) from a human temporal cortex library. This 2.5-kb cDNA encodes a 689-amino acid protein with a predicted molecular mass of approximately 78.5 kDa. The primary structure of h-MP78 exhibits high similarity to those of porcine (94%) and rat (92%) thimet oligopeptidase. Expression of the cDNA in HEK-293 resulted in the production of an active enzyme able to cleave a chromogenic beta-APP derived substrate peptide KTEEISEVKM-P-nitro-anilide. RNA blot analysis of various human tissues revealed one major species of h-MP78 mRNA of approximately 2.55 kb. The highest level of mRNA was found in the brain.