The aim of this study was to quantify the expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vascular endothelial cells (HUVECs) exposed to anoxia/reoxygenation (A/R) in the presence or absence of an inflammatory context (0.1 IU/ml tumor necrosis factor-alpha [TNF-alpha]) and to investigate the effects of two different NADPH inhibitors, apocynin and diphenyleneiodonium (DPI), on the expression of the endothelial cell adhesion molecules. Confluent HUVECs were exposed to anoxia for 3 hours (100% N2), followed by a reoxygenation period of 4 hours. TNF-alpha at 0.1 IU/ml was added to the medium either under normoxic conditions for 7 hours (TNF-alpha) or just before the start of anoxia (A/R + TNF-alpha). Levels of E-selectin, VCAM-1, and ICAM-1 were quantified using specific monoclonal antibodies revealed by an alkaline phosphatase-labeled goat F(ab)'2 fragment against mouse IgG antibody and the fluorescent substrate Attophos. Adhesion experiments were also performed using calcein-labeled U937 leukocytes. HUVECs submitted to A/R overexpressed E-selectin but not VCAM-1 or ICAM-1, whereas TNF-alpha at 0.1 IU/ ml increased the expression of all three adhesion molecules. In endothelial cells subjected to A/R in the presence of TNF-alpha, a synergistic increase of E-selectin expression and a synergistic adhesion of U937 cells was noted. The NADPH oxidase inhibitors apocynin and DPI both decreased significantly the U937 adhesion and the E-selectin overexpression on HUVECs submitted to A/R, TNF-alpha, or A/R + TNF-alpha. These results suggest that E-selectin expression is implicated in the leukocyte adhesion to HUVECs caused by A/R in the presence or absence of an inflammatory context. NADPH oxidase appears to participate in the E-selectin overexpression on HUVECs subjected either to A/R and/or TNF-alpha, suggesting a major role of this enzyme in the ischemia/reperfusion syndrome.