Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The pro-inflammatory cytokine TNF causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. NO synthase and xanthine oxidase inhibitors (L-NNA and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, TTFA and rotenone) attenuated TNF-induced superoxide (O₂^.-) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-evoked O₂^.- generation and apoptosis. TNF rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O₂^.- production, and accounts for a burst of oxidative stress in response to TNF. Nox4siRNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF in CMVEC. Nox4 is co-localized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O₂^.- and apoptosis caused by TNF stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-induced oxidative stress by inhibiting Nox4 activity and/or by O₂^.- scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease.