Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) are positive regulators of cardiac hypertrophy, but the nature of cross-talk between CaMKII and CaN signaling pathways in hypertrophic cardiomyocytes remains unclear. Here we documented that CaMKIIdelta3 activation enhances transcription of the CaN gene through activation of the CaN-Abeta subunit (CnAbeta) promoter in rat cultured cardiomyocytes. Co-immunoprecipitation assays showed that MEF2 forms a complex with GATA4 following transfection of an active CaMKIIdelta3 (T278D) mutant in neonatal cardiomyocytes. Inversely, transfection of a dominant negative CaMKIIdelta3 mutant failed to promote a MEF2-GATA4 complex. Consistent with these observations, immunocytochemistry indicated nuclear co-localization of MEF2 with GATA4 after hypertrophic agonist stimulation or CaMKIIdelta3 (T278D) transfection. These data demonstrate that CaMKII can enhance CnAbeta promoter activity by enhancing MEF2-GATA4 synergy, suggesting a novel mechanism for CaMKII-mediated hypertrophic signaling, which contributes to induction and development of the hypertrophic response through CaN activation.