Brief episodes of ischemia can render an organ resistant to subsequent severe ischemia. This ‘ischemic preconditioning’ is ascribed to various mechanisms, including oxidative stress. We investigated whether preconditioning exists on an endothelial level. Human umbilical vein endothelial cells (HUVECs) were transiently confronted with oxidative stress (1 mM H₂O₂, 5 min). Adhesion molecules ICAM‐1 and E‐selectin and release of cytokines IL‐6 and IL‐8 to subsequent stimulation with TNF‐α (2.5 ng/ml, 4 h) were measured (flow cytometry and immunoassay), as were nuclear translocation of the transcription factor NFκB (Western blotting, confocal microscopy) and redox status of HUVECs (quantification of glutathione by HPLC). TNF‐α elevated IL‐6 in the cell supernatant from 8.8 ± 1to41 ± 3 pg/ml and IL‐8 from 0.5 ± 0.03 to 3 ± 0.2 ng/ml. ICAM‐1 was increased threefold and E‐selectin rose eightfold. Oxidative stress (decrease of glutathione by 50%) reduced post‐TNF‐α levels of IL‐6 to 14 ± 3 and IL‐8 to 1 ± 0.2; the rise of ICAM‐1 was completely blocked and E‐selectin was only doubled. The anti‐inflammatory effects of preconditioning via oxidative stress were paralleled by reduction of the translocation of NFκB on stimulation with TNF‐α, and antagonized by the intracellular radical scavenger N‐acetylcysteine. ‘Anti‐inflammatory preconditioning’ of endothelial cells by oxidative stress may account for the inhibitory effects of preconditioning on leukocyte adhesion in vivo.—Zahler, S., Kupatt, C., Becker, B. F. Endothelial preconditioning by transient oxidative stress reduces inflammatory responses of cultured endothelial cells to TNF‐α. FASEB J. 14, 555–564 (2000)