We report here that cultured human lymphoma cells in the absence of sonicated eosinophils are sensitive to killing by glucose oxidase (β-d-glucose:oxygen-oxido reductase; EC 1.1.3.4) at concentrations as low as 0.025 µg/ml, a level that can be rapidly attained in s.c. tumor implants in mice that receive a single nonlethal injection of enzyme. Multiple clonogenic assays were used to measure the survival of human lymphoma cell lines (H9 and ARH-77) cultured for 14 days in complete RPMI 1640 supplemented with exogenous glucose oxidase (0.025–2.5 µg/ml) or an immunoconjugate containing glucose oxidase (0.25–25 µg/ml) in the presence or absence of catalase (10 µg/ml) or an equal number of sonicated human eosinophils with or without supplemental 100 µm Br^-, I^-, or SCN^-. In addition, we used an immunoassay to measure the concentration of glucose oxidase in s.c. implants of the Sp 2/0 myeloma tumor at 0–30 min after an i.v. injection of 50 µg of enzyme into 21 BALB/c mice. Doses of glucose oxidase as small as 0.025 µg/ml killed more than 3 logs of tumor cells. Catalase completely inhibited, and sonicated human eosinophils partially inhibited, the killing by glucose oxidase or immunoconjugate, whereas supplemental halides had no effect. Glucose oxidase i.v. produced levels >0.04 µg/g of tumor for 30 min after injection with a peak concentration of 0.079 µg/g of tumor within 5 min of injection. These results are important because certain human lymphomas contain extensive extracellular deposits of eosinophil peroxidase, thereby making these tumors potentially less susceptible to killing by otherwise therapeutic doses of glucose oxidase.