Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO 2 Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480±198 μmol/mol tyrosine; n= 11) compared with nonasthmatic subjects (52.5±40.7 μmol/mol tyrosine; n= 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)-and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093±457 μmol BrY/mol tyrosine; 161±88 μmol ClY/mol tyrosine; n= 11 each) compared with nonasthmatic subjects (13±14.5 μmol BrY/mol tyrosine; 65±69 μmol ClY/mol tyrosine; n= 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO 2 Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO 2−, NO 3−, or NO 2 Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO 2− or an exogenous NO source. At low, but not high (eg,> 2 μM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.