Tyrosine nitration is considered a key reaction of peroxynitrite‐triggered tissue injury in inflammatory diseases. We investigated the potential involvement of peroxynitrite in protein tyrosine nitration in isolated murine peritoneal macrophages activated either in vitro with interferon‐γ/lipopolysaccharide or in vivo by priming mice with Corynebacterium parvum (10 mg×kg^‐1). Both protocols led to release of NO and accumulation of nitrite accompanied by formation of protein‐bound 3‐nitrotyrosine. Oxidation of dihydror‐hodamine 123, a measure of peroxynitrite release, remained close to basal levels upon in vitro activation of the macrophages but was increased ~twofold in vivo. Tyrosine nitration in macrophages activated in vitro was inhibited by catalase and the time course of nitration correlated with nitrite accumulation, whereas superoxide (O₂^•–) and H₂O₂ release occurred at much earlier times. To address the contribution of O₂*^‐ and peroxynitrite to in vivo nitration, aO₂^•– scavenger (MnTBAP;1 mg×kg^‐1) was given to C. parvum‐primed mice. MnTBAP led to almost complete inhibition of C. parvum‐triggered O₂^•– and peroxynitrite release, whereas nitrite accumulation and formation of 3‐nitro‐tyrosine were less affected (~50% of controls). These results argue against an essential role of peroxynitrite in protein tyrosine nitration in vivo.—Pfeiffer, S., Lass, A., Schmidt, K., Mayer, B. Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo: evidence against an essential role of peroxynitrite. FASEB J. 15, 2355–2364 (2001)