NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions. Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-alpha. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF-alpha-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67(phox), p47(phox), p22(phox)). Inhibitors of AP-1 significantly diminished the Ang II or TNF-alpha-stimulated p22(phox) promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22(phox) promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1-binding sites in the p22(phox) gene promoter. In SMCs exposed to Ang II or TNF-alpha, inhibition of AP-1-related pathways reduces NADPHox expression and the O(2)(-) production. The physical interaction of AP-1 with p22(phox) gene promoter facilitates NADPHox regulation.