A cDNA clone encoding a human NK serine protease was obtained by screening a lambda-gt10 library from the Lopez NK leukemia with the rat natural killer Met-ase (RNK-Met-1) cDNA clone. In Northern blot analysis human Met-ase (Hu-Met-1) cDNA hybridized with a 0.9-kb mRNA in two human NK leukemia cell lines, unstimulated human PBMC, and untreated purified CD3-CD56+ large granular lymphocytes. Unlike other members of the granzyme family that are highly expressed in activated peripheral T cells, the Hu-Met-1 transcript was barely detected in a population of PMA and ionomycin or IL-2-treated high density T cells. Several in vitro cultured Burkitt lymphomas, chronic- and promyeloid leukemias, acute lymphoblastic leukemias, and colon and ovarian carcinomas and colon and ovarian carcinomas did not express Hu-Met-1 mRNA. Hu-Met-1 mRNA expression in a small number of human T cell tumor lines did not correlate with any particular phenotype or stage of development. The presence of Hu-Met-1 mRNA closely correlated with the Met-ase activity of cellular lysates prepared from these various human peripheral blood subsets and in vitro cultured cell lines. Met-ase activity detected in whole cell lysates of cytotoxic lymphocytes was associated with the cytoplasmic granules of these cells. The nucleotide sequence of the Hu-Met-1 cDNA clone encodes a predicted serine protease of 257 amino acids. The predicted protein is an active enzyme of 232 amino acids with a calculated unglycosylated m.w. of 27,100. Hu-Met-1 is 66% identical to RNK-Met-1 at the amino acid level. The human and rat mature protein sequences conserve the active site His, Asp, and Ser amino acids that form the catalytic triad of serine proteases, all 8 cysteine residues, and several amino acids critical in the formation of the substrate binding pocket. The gene for the Hu-Met-1 serine protease is located on chromosome 19, which distinguishes it from any other member of the human granzyme family.