Eicosanoid production is reduced when the nitric oxide (NO^.) pathway is inhibited or when the inducible NO^. synthase gene is deleted indicating that the NO^. and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO^. and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100 µM - 500 µM) and SIN-1 (200 µM) stimulate phosphorylation of the extracellular signal-regulated kinase (ERK), p38 and cytosolic phosholipase A₂ (cPLA₂). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. Using the p38 inhibitor SB20219, it was demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release while U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of the Ca²⁺/calmodulin dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Taken together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA₂ activation and arachidonic acid release. Thus, studies investigating the role of the NO^. pathway on eicosanoid production need to consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease.