Abstract: Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor‐α (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF‐mediated EC activation. EC apoptosis is likely important in the process termed “plaque erosion” that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide‐dependent protective pathways that involve enzymes PI3‐kinase and Akt. Conversely, our laboratory has identified nitric oxide‐independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen‐activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF‐mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm²) on TNF‐stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c‐Jun N‐terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre‐exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen‐activated protein kinase kinase inhibitor, blocked the flow‐mediated inhibition of TNF activation of JNK. Flow‐mediated inhibition of JNK was unaffected by 0.1 mM L‐nitroarginine, 100 μM 8‐bromo‐cyclic GMP, or 100 μM 8‐bromo‐cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow‐mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.