Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is expressed in many mammalian cells, with the delta isoform predominantly expressed in cardiomyocytes. Previous studies have shown that inhibition of CaMKII protects cardiomyocytes against beta(1)-adrenergic receptor-mediated apoptosis. However, it is unclear whether activation of CaMKII is sufficient to cause cardiomyocyte apoptosis and whether CaMKII signaling is important in heart muscle cell apoptosis mediated by other stimuli. Here, we specifically enhanced or suppressed CaMKII activity using adenoviral gene transfer of constitutively active (CA-CaMKII(deltaC)) or dominant negative (DN-CaMKII(deltaC)) mutants of CaMKII(deltaC) in cultured adult rat cardiomyocytes. Expression of CA-CaMKII(deltaC) promoted cardiomyocyte apoptosis that was associated with increased mitochondrial cytochrome c release and attenuated by co-expression of Bcl-X(L). Importantly, isoform-specific suppression of CaMKII(deltaC) with the DN-CaMKII(deltaC) mutant similar to nonselective CaMKII inhibition by the pharmacological inhibitors (KN-93 or AIP) not only prevented CA-CaMKII(deltaC)-mediated apoptosis but also protected cells from multiple death-inducing stimuli. Thus, activation of CaMKII(deltaC) constitutes a common intermediate by which various death-inducing stimuli trigger cardiomyocyte apoptosis via the primary mitochondrial death pathway.