A previously identified cDNA encoding a human gamma-glutamyl hydrolase was expressed in a baculovirus system. The expressed protein had molecular mass of 37 kDa. Treatment of the protein with PNGase F produced a protein of molecular mass of 30 kDa, indicating that the protein contained asparagine-linked glycosylation. Sequence analysis of the expressed protein indicated that a 24-amino-acid signal peptide had been removed. A polyclonal antibody to the expressed enzyme was used in Western blot analysis of partially purified lysates of HL-60 promyeloid leukemia cells and MCF-7 breast cancer cells. The HL-60 and MCF-7 enzymes appeared as two closely spaced bands with a molecular mass of 37 kDa. Treatment of the HL-60 enzyme with PNGase F produced a protein with a molecular mass of 30 kDa. The activities of the expressed enzyme and the enzyme from HL-60 cells were similar on methotrexate polyglutamates. Methotrexate-gamma-Glu is a poor substrate for the human enzyme relative to methotrexate gamma-Glu2-5. During hydrolysis of methotrexate-gamma-Glu4, all possible pterin-containing cleavage products (methotrexate and methotrexate-gamma-Glu1-3) appear. The results demonstrated that the human enzyme cleaves both the ultimate and penultimate gamma-linkages of methotrexate polyglutamates. Glutamate was released as either glutamic acid or gamma-Glu2. Longer chain species of gamma-Glun>2 were not observed. Inhibition by iodoacetic acid suggested that both the expressed enzyme and the HL-60 enzyme may contain a catalytically essential cysteine. These results indicate that the identified cDNA encodes the intracellular gamma-glutamyl hydrolase found in a variety of human tumor cells and that the baculovirus-expressed enzyme is a suitable model for further structural and enzymatic studies.