The in vitro testing of antitumor drugs involves the use of mouse and human tumor cells. In particular, there is interest in developing agents active against human solid tumors. We examined several biochemical parameters that may contribute to the differential sensitivity of the cell lines used in our laboratory to the toxic effects of antitumor compounds. The tumor cell lines examined were of mouse (colon 38, L1210 leukemia, and C1498 leukemia) and human origin (CEM leukemia, CX1 colon, H116 colon, HCT8 colon and H125 lung). Quinone reductase activity was markedly different between leukemia and solid-tumor cell lines of either mouse or human origin, with increased activity being observed in the solid-tumor cell lines relative to the leukemia lines. GSH transferase activity also was generally increased in solid-tumor relative to leukemia cell lines. Superoxide dismutase activity and thiol levels were similar in leukemia and solid-tumor cell lines, except that thiol levels were very low in colon 38. Mouse cell lines from in vitro passage had somewhat higher activity of superoxide dismutase and thiol levels than did cells maintained in vivo, indicating relatively increased antioxidant defenses. The toxicity of 2,3-dimethoxy-1,4-naphthoquinone, a model quinone that exerts its toxic effects via production of reactive oxygen species, was significantly lower in mouse lines maintained in vitro than in those tested in vivo, whereas the toxicity of another quinone, menadione, was just slightly lower. Quinone reductase activity, GSH transferase activity, and thiol levels were significantly higher in the human lines than in the mouse lines. Accordingly, the toxicity of both quinones tended to be lower in the human lines than in the mouse lines.