Shear stress due to blood flow is a major determinant of endothelial function. Generally, sustained laminar shear (LS) leads to endothelial NO synthase(eNOS)-derived nitric oxide (NO) production and is vasodilator, anti-inflammatory and anti-atherogenic. However, sudden increases in LS trigger superoxide production for the first minutes via endothelial NADPH oxidase complex. Recently, we showed in vascular smooth muscle cells that NADPH oxidase is regulated by the thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to be S-nitrosated by NO. We hypothesized that PDI-dependent support of NADPH oxidase activity may affect NO output during sustained LS. Cultured rabbit aortic endothelial cells (RAEC) were submitted to LS (15 dynes/cm²) in a cone-plate system. After 18h, as compared with static controls, RAEC exhibited: a) decreased (~50%) superoxide production (HPLC analysis of DHE oxidation); b) decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay); c) increased (~10%) SOD activity (inhibition of cyt.-c reduction); d) Increased eNOS expression (~20%, western blot); e) increased nitrite levels in culture medium (2-fold, NO Analyzer and Griess reaction). Intriguingly, analysis of NADPH oxidase activity in membrane fraction through DHE oxidation, usually employed in many cell types, showed 4 to 6-fold paradoxical increase, partially (30%) prevented by eNOS inhibitor L-NMMA (100mM). Thus, such assay appears heavily contaminated by artificial in vitro eNOS uncoupling in membrane fractions from RAEC submitted to LS. Total PDI expression was unchanged after 18h of LS, while protein expression of GRP78, a chaperone associated with endoplasmic reticulum stress, decreased (~30%). The role of PDI in NO output was investigated through gain or loss-of-function experiments with transfection of sense or antisense PDI cDNA, the later known to decrease superoxide levels and NADPH oxidase actvity in other cells. While PDI-sense transfected cells submitted to LS showed slight decrease in nitrite levels, PDI-antisense led to ~50% increase in nitrite output. In static RAEC, PDI-antisense decreased NADPH oxidase activity ~15%. These results suggest that PDI is involved in regulation of NO output during LS and raise the hypothesis that PDI effect involves a balance between its S-nitrosation and support of NADPH oxidase activity.(Supported by FAPESP and CNPq Milênio Redoxoma)