Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deanunase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was iinjected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein.The recombinant human adenosine deaninase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.Adenosine deaminase (ADA; adenosine-aminohydrolase, EC 3.5.4.4) catalyzes the conversion ofadenosine and deoxyadenosine to inosine and deoxyinosine. In humans deficiency of this purine salvage enzyme is associated with severe immunodeficiency and lymphopenia in both B-and T-cell lineages (1, 2 known about the enzymatic activity of human ADA, including its interaction with various substrates (4) and inhibitors (6-8), no direct information is available concerning the enzyme active site. Although a preliminary analysis of crystals of murine ADA has been reported (9), the three-dimensional fine structure of the human enzyme is not known.It has been suggested that the enzyme undergoes a conformational change upon inhibitor binding (6,7) and that a reactive histidine group participates in catalysis of calf intestinal ADA (10). However, lack of sufficient quantities of human ADA has precluded detailed structural studies of important enzyme domains.Human thymus contains relatively abundant quantities of ADA (7), but the scarcity of this tissue has rendered impractical the purification of the large quantities of protein required for structural and mechanistic studies. We have therefore explored production of ADA ...