Alveolar macrophages, which generate high levels of reactive oxygen species, especially O2•-, are involved in the recruitment of neutrophils to sites of inflammation and injury in the lung, and the generation of chemotactic proteins triggers this cellular recruitment. In this study, we asked whether O2•- generation in alveolar macrophages had a role in the expression of chemokines. Specifically, we hypothesized that O2•- generation is necessary for chemokine expression in alveolar macrophages after TNF-α stimulation. We found that alveolar macrophages have high constitutive NADPH oxidase activity that was not increased by TNF-α, but TNF-α increased the activity of the mitochondrial respiratory chain. In addition, the mitochondrial respiratory chain increased O2•- generation if the NADPH oxidase was inhibited. O2•- generation was necessary for macrophage inflammatory protein-2 (MIP-2) gene expression, because inhibition of NADPH oxidase or the mitochondrial respiratory chain or overexpression of Cu,Zn-superoxide dismutase significantly inhibited expression of MIP-2. TNF-α activated the ERK MAP kinase, and ERK activity was essential for chemokine gene expression. In addition, overexpression of the MEK13ERK pathway significantly increased IL-8 expression, and a small interfering RNA to the NADPH oxidase inhibited ERK- and TNF-α-induced chemokine expression. Collectively, these results suggest that in alveolar macrophages, O 2•- generation mediates chemokine expression after TNF-α stimulation in an ERK-dependent manner.