In the literature, biological tyrosine nitrations have been reported to depend not only on peroxynitrite but also on nitrite/hydrogen peroxide linked to catalysis by myeloperoxidase. In endotoxin-stimulated RAW 264.7 macrophages, we have detected a major nitrotyrosine positive protein band around 72 kDa and identified it as prostaglandin endoperoxide synthase-2 (PGHS-2). Isolated PGHS-2 in absence of its substrate arachidonate was not only tyrosine-nitrated with peroxynitrite, but also with nitrite/hydrogen peroxide in complete absence of myeloperoxidase. Our data favor an autocatalytic activation of nitrite by PGHS-2 with a subsequent nitration of the essential tyrosine residue in the cyclooxygenase domain. Under inflammatory conditions, nitrite formed via NO-synthase-2 may therefore act as an endogenous regulator for PGHS-2 in stimulated macrophages. Nitration of PGHS-2 by the autocatalytic activation of nitrite further depends on the intracellular concentration of arachidonate since arachidonate reacted competitively with nitrite and could prevent PGHS-2 from nitration when excessively present.