Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H 2 O 2. Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H 2 O 2, but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H 2 O 2 formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H 2 O 2 to nitrogen dioxide radical (NO 2•) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS–DUOX2–TPO–NO 2• metabolome drives nitrative stress in HAEC and likely in SA.