Abstract —Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox ^{− /−} , and endothelial NO synthase (eNOS) ^{− /−} mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS ^{− /−} mice, but was attenuated in segments from gp91phox ^{− /−} mice. Endothelium-dependent relaxation was greater in aortae from gp91phox ^{− /−} than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox ^{− /−} mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO. ( Circ Res. 2000;87:26-32.)