—Increased steady intraluminal pressure in blood vessels activates the extracellular signal–regulated kinase (ERK)1/2 pathway. However, signal transduction of pulsatile stretch has not been elucidated. Using an organ culture model of rabbit aorta, we studied ERK1/2 activation by pulsatility in vessels maintained at 80 mm Hg for 24 hours. ERK1/2 activity was evaluated by in-gel kinase assays and by Western blot. Compared with control aortas without pulsatility, aortas submitted to a pulsatile 10% variation in vessel diameter displayed a significant increase in ERK1/2 activity (207±12%, P<0.001), which remained high after removal of the endothelium. Unlike steady overstretch, pulsatile stretch–induced activation of ERK1/2 was not modified by herbimycin A, a Src family tyrosine kinase inhibitor, but was reduced by other tyrosine kinase inhibitors, tyrphostin A48 and genistein (162±27% and 144±14%, respectively). Conversely, ERK1/2 activity was markedly decreased in pulsatile vessels treated with staurosporine (114±18%) although neither of the more specific protein kinase C inhibitors, Ro-31-8220 or Gö-6976, blocked ERK1/2 activation (209±24% and 238±34%, respectively), whereas staurosporine had no effect on steady overstretch–induced ERK1/2 activation. Pulsatility induced superoxide anion generation, which was prevented by the NADPH oxidase inhibitor diphenyleneiodonium. Furthermore, polyethylene glycol–superoxide dismutase completely abolished ERK1/2 activation by pulsatility (114±12%). Finally, ERK1/2 and O₂⁻ levels in freshly isolated vessels were equivalent to the levels found in pulsatile vessels. In conclusion, pulsatile stretch activates ERK1/2 in the arterial wall via pathways different from those induced by steady overstretch. Pulsatility might be considered a physiological stimulus that maintains a certain degree of ERK1/2 activation via oxygen-derived free radical production.