Because reactive oxygen species (ROS) can function as second messengers and regulate the activation of the transcription factor nuclear factor (NF)-kappaB, we investigated the possible role of tumor necrosis factor-alpha (TNF-alpha)-induced ROS generation in endothelial cells in signaling E-selectin gene transcription. We demonstrated that stimulation of human pulmonary artery endothelial cells with TNF-alpha (100 U/ml) resulted in ROS production using the oxidant-sensitive dye 5 (and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester. Pretreatment with N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) for 0.5 h inhibited TNF-alpha-induced generation of ROS as well as activation of NF-kappaB and E-selectin mRNA and the cell surface protein expression. These findings indicate that TNF-alpha induces NF-kappaB activation and the resultant E-selectin gene expression by a pathway that involves formation of ROS and that E-selectin expression can be inhibited by the antioxidant action of NAC or PDTC. The results support the hypothesis that generation of ROS in endothelial cells induced by proinflammatory cytokines such as TNF-alpha is a critical signal mediating E-selectin expression.