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Objective-Despite studies implicating superoxide anion-producing oxidases in atherosclerosis, their characteristics, expression, and regulation in cells of lesions are poorly understood. We examined the following: (1) whether cytochrome b 558 -dependent NAD(P)H oxidase-phox peptides are expressed by intimal smooth muscle cells (iSMCs) and macrophages of human aortic atherosclerotic lesions and their regulation and (2) whether cytochrome b 558 -dependent NAD(P)H oxidase represents a major NAD(P)H oxidase in iSMCs. Methods and Results-Using a combination of immunochemical and reverse transcription-polymerase chain reaction procedures, we demonstrate that p22 phox and gp91 phox (cytochrome b 558 ) expression in normal intima was restricted to a quarter of the iSMCs. In fatty streaks, a similar fraction of iSMCs expressed cytochrome b 558 , whereas macrophages also expressed low levels of p47 phox and p67 phox . In fibrofatty lesions, the majority of iSMCs expressed the cytochrome b 558 subunits; p67 phox was also detected. Macrophages and macrophage-derived foam cells expressed the 4 phox subunits that constitute superoxide-producing cytochrome b 558 -dependent NAD(P)H oxidase. These were upregulated by transforming growth factor-␤ 1 and interferon-␥. Aortic lesions also expressed Thox1 and Nox4, and although their expression also increases with lesion severity, their expression is less frequent than that of gp91 phox . Conclusions-In human aortic fibrofatty lesions, a cytochrome b 558 -dependent NAD(P)H oxidase appears to be a major iSMC and macrophage oxidase whose expression is upregulated by cytokines. (Arterioscler Thromb Vasc Biol. 2002;