Oxysterols are common components of oxidized low-density lipoprotein and accumulate in the core of fibrotic plaques as a mixture of cholesterol and cholesteryl ester oxidation products. The proapoptotic effects of a biologically representative mixture of oxysterols was compared with equimolar amounts of 7-ketocholesterol and unoxidized cholesterol. The oxysterol mixture in a concentration range actually detectable in hypercholesterolemic patients did not stimulate programmed cell death in cultivated murine macrophages. Unoxidized cholesterol also produced no effect. By contrast, when given alone, 7-ketocholesterol strongly stimulated the mitochondrial pathway of apoptosis with cytochrome c release, caspase-9 activation, and eventually caspase-3 activation. Subsequent experiments showed that when 7-ketocholesterol was administered to cells together with another oxysterol, namely 7betaOH-cholesterol, the strong proapoptotic effect of 7-ketocholesterol was markedly attenuated. As regards the mechanism underlying this quenching, we found that the combined oxysterol treatment counteracted the ability of 7-ketocholesterol, when administered alone, to strongly up-regulate the steady-state levels of reactive oxygen species (ROS) without interfering with sterol uptake. Furthermore, this increase in intracellular ROS appeared to be responsible for the up-regulation of proapoptotic factor, p21, after treatment with 7-ketocholesterol but not in cells challenged with the oxysterol mixture. Competition among oxysterols, apparently at the level of NADPH oxidase, diminishes the ROS induction and direct toxicity that is evoked by specific oxysterols. As a consequence, a more subtle gene modulation by oxysterols becomes facilitated in vascular cells.