Hepsin, a putative membrane-bound serine protease, was originally identified as a human liver cDNA clone (Leytus, S.P., Loeb, K.R., Hagen, F.S., Kurachi, K., and Davie, E.W. (1988) Biochemistry 27, 1067-1074). In the present study the human hepsin gene was localized to chromosome 19 at q11-13.2. The messenger RNA of hepsin is 1.85 kilobases in size and present in most tissues, with the highest level in liver. Hepsin is synthesized as a single polypeptide chain, and its mature form of 51 kDa was found in various mammalian cells including HepG2 cells and baby hamster kidney cells. It is present in the plasma-membrane in a molecular orientation of type II membrane-associated proteins, with its catalytic subunit (carboxyl-terminal half) at the cell surface, and its amino terminus facing the cytosol. Hepsin is found neither in cytosol nor in culture media. The results obtained suggest that hepsin has an important role(s) in cell growth and function.