Nitration of tyrosine residues in proteins occurs in a wide range of inflammatory diseases involving neutrophil and macrophage activation. We report that both myeloperoxidase (MPO) and horseradish peroxidase (HRP) utilize nitrite (NO₂⁻) and hydrogen peroxide (H₂O₂) as substrates to catalyze tyrosine nitration in proteins. MPO was approximately 10 times more effective than HRP as a nitration catalyst of bovine serum albumin (BSA). Nitration of BSA by MPO did not require chloride as a cofactor. Physiologic levels of chloride did not significantly inhibit nitration by MPO. Oxidation of chloride to hypochlorous acid (HOCl) is catalyzed by MPO but not by HRP, yet HRP also catalyzed nitration from hydrogen peroxide plus nitrite. Therefore, HOCl formation was not obligatory for tyrosine nitration. Although HOCl plus nitrite can nitrate the amino acid tyrosine in simple solutions, protein nitration by HOCl plus nitrite was not observed in heart homogenates, probably due to the presence of multiple alternative targets of both HOCl and HOCl plus nitrite. In contrast, MPO catalyzed nitration of many proteins in rat heart homogenates using NO₂⁻plus H₂O₂, suggesting that peroxidase-catalyzed nitration of tyrosine could occur in the presence of competing substratesin vivo.HOCl could substitute for H₂O₂as the oxidizing substrate for nitration of either BSA or tissue homogenates catalyzed by either peroxidase. Activated neutrophils may generate nitrotyrosine by several mechanisms, including peroxynitrite, HOCl plus nitrite, and a chloride-independent mechanism involving MPO, nitrite, and hydrogen peroxide.