The phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is a multiprotein enzyme that catalyzes the production of microbicidal oxidants. Although oxidase assembly involves association of several membrane and cytosolic oxidase proteins, one of the cytosolic cofactors, p67(phox), appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67(phox), we investigated transcriptional regulation of the p67(phox) gene [neutrophil cytosol factor 2 (NCF2)] and demonstrated previously that activated protein-1 (AP-1) was essential for basal transcriptional activity. As p67(phox) can be up-regulated by tumor necrosis factor alpha (TNF-alpha), which activates AP-1, we hypothesized that TNF-alpha might regulate NCF2 transcription via AP-1. In support of this hypothesis, we show here that NCF2 promoter-reporter constructs are up-regulated by TNF-alpha but only when AP-1 factors are coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67(phox) protein are up-regulated by TNF-alpha in various myeloid cell lines as well as in human monocytes. It is surprising that mutagenesis of the AP-1 site in NCF2 promoter constructs did not eliminate TNF-alpha induction, suggesting additional elements were involved in this response and that AP-1 might play a more indirect role. Indeed, we used NCF2 promoter-deletion constructs to map a novel TNF-alpha-responsive region (TRR) located between -56 and -16 bp upstream of the translational start site and demonstrated its importance in vivo using transcription factor decoy analysis. Furthermore, DNase footprinting verified specific binding of factor(s) to the TRR with AP-1 binding indirectly to this region. Thus, we have identified a novel NCF2 promoter/enhancer domain, which is essential for TNF-alpha-induced up-regulation of p67(phox).