The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (·O2−) formation. In this study, we analysed the effect of laminar shear stress on ·O2− formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced ·O2− formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm−2, 24 h) down-regulated ·O2− formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91phox and p47phox. In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of ·O2− formation, gp91phox and p47phox expression by long-term laminar shear stress was blocked by l-NAME. NO donor DETA-NO down-regulates ·O2− formation, gp91phox and p47phox expression in static cultures. In conclusion, our data suggest a transient activation of ·O2− formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91phox/p47phox-containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/·O2− balance and the vasoprotective potential of physiological levels of laminar shear stress.