Macrophages are involved in the pathogenesis of immune and inflammatory disorders of the lung. Tumor necrosis factor (TNF)-α is a proinflammatory cytokine that has been linked to both injury and repair in the acute and chronic lung diseases. TNF-α also generates reactive oxygen species, including superoxide anion. We have previously shown that reactive oxygen species (ROS) generation is important in macrophage cytokine gene expression, so we hypothesized that superoxide anion generation was necessary for MIP-2 expression after TNF-α stimulation. Using real time PCR, we found that TNF-α increased MIP-2 expression greater than 25-fold, and cells exposed to diphenyleneiodonium (DPI), a non-specific inhibitor of NADPH oxidase, decreased expression to control levels. To assure ourselves that the effect of DPI was due to decreased superoxide anion, we performed similar experiments with apocynin, a specific NADPH oxidase inhibitor, and L-NMMA, an inhibitor of nitric oxide synthase. We found that apocynin inhibited MIP-2 expression approximately 50%, and L-NMMA had no significant effect in macrophages stimulated with TNF-α. To further confirm our hypothesis, we over expressed Cu,Zn-SOD and found that MIP-2 expression was reduced to near control levels when compared to cells expressing an empty vector. These findings show that MIP-2 gene expression in TNF-α-stimulated macrophages is dependent on adequate generation of superoxide anion and is only partially due to NADPH activity. VA Merit Review.