The increased expression of immediate-early genes is a key feature of the myocardial response to hypertrophic stimuli. In this study, we investigated whether pressure overload or phenylephrine treatment stimulated myocyte enhancer factor 2 (MEF2)-dependent transcriptional activation of c-jun in cardiac myocytes. Western blotting and immunohistochemical analysis of rat myocardium demonstrated that p70MEF2 is highly expressed in the rat heart and is predominantly located at the nuclei of cardiac myocytes. Electrophoretic mobility shift assays of myocardial nuclear extracts revealed a consistent DNA binding activation of MEF2 after 1 and 2 hours of pressure overload. We further showed that pressure overload induced a progressive nuclear translocation and activation of extracellular signalregulated kinase 5 (ERK5). Coimmunoprecipitation and in vitro kinase assays indicated that the activation of ERK5 was paralleled by increased association of ERK5/p70MEF2 and by enhanced ability of ERK5 to phosphorylate p70MEF2. Experiments with in vivo transfection of the left ventricle with the c-jun promoter reporter gene showed that pressure overload induced a consistent increase of c-jun transcriptional activity in the rat myocardium. Rendering the MEF2 site of the c-jun plasmid inactive by mutation abolished the load-induced activation of the c-jun promoter reporter gene. Mutation of the MEF2 site also abolished the phenylephrine-induced c-jun promoter activation in neonatal rat ventricular myocytes. In addition, we demonstrated that neonatal rat ventricular myocyte transfection with ERK5antisense oligodeoxynucleotide inhibited the phenylephrine-induced c-jun promoter activation. 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