The cytotoxicity of the oxysterols 25-hydroxycholesterol, 7 beta -hydroxycholesterol, cholesterol-5 alpha ,6 alpha -epoxide, cholesterol-5 beta ,6 beta -epoxide, 19-hydroxycholesterol and 7-ketocholesterol was examined in U937 cells, a human monocytic blood cell line. 7 beta -Hydroxycholesterol, cholesterol-5 beta ,6 beta -epoxide, and 7-ketocholesterol, at 30 mu mol/L concentration, were found to be cytotoxic to this cell line and the mode of cell death was by apoptosis. 25-Hydroxycholesterol, cholesterol-5 alpha ,6 alpha -epoxide and 19-hydroxycholesterol (30 mu mol/L) did not induce apoptosis in this cell line. Since it has been suggested that the generation of an oxidative stress may occur in the early stages of the apoptotic process, the glutathione concentration and the activity of superoxide dismutase were also measured in the oxysterol-treated cells. 7 beta -Hydroxycholesterol was shown to increase the superoxide dismutase activity and decrease the glutathione concentration. However, cholesterol-5 beta ,6 beta -epoxide and 7-ketocholesterol, which were also shown to induce apoptosis, did not affect the glutathione concentration or the superoxide dismutase activity in the U937 cells. Therefore, oxysterol-induced apoptosis may not be dependent on the generation of an oxidative stress.