Atherosclerosis is considered as a chronic inflammatory process. However, the nature of the oxidant signaling that regulates monocyte adhesion and its underlying mechanism is poorly understood. We investigated the role of reactive oxygen species on the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion in the cultured endothelial cells. TNF-α at a range of 1~30 ng/ml induced VCAM-1 expression dose-dependently. BCECF-AM-labeled U937 cells firmly adhered on the surface of endothelial cells when the endothelial cells were incubated with TNF-α (15 ng/ml). Ten μmol/L of SB203580, an inhibitor of p38 MAPK, significantly reduced TNF-α-induced VCAM-1 expression, compared to the JNK inhibitor (40μmol/L of SP60015) or ERK inhibitor (40μmmol/L of U0126). Also, SB203580 significantly inhibited TNF-α-induced monocyte adhesion in HUVEC. Superoxide production was minimal in the basal condition, however, treatment of TNF-α induced superoxide production in the dihydroethidine- loaded endothelial cells. Diphenyleneiodonium (DPI, 10μmol/L), an inhibitor of NADPH oxidase, and rotenone (1μmol/L), an inhibitor of mitochondrial complex I inhibited TNF-α-induced superoxide production, VCAM-1 expression and monocyte adhesion in the endothelial cells. Taken together, our data suggest that NADPH oxidase and mitochondrial ROS were involved in TNF-α-induced VCAM-1 and monocyte adhesion in the endothelial cells.