Recent studies have demonstrated that transgenic (TG) expression of either Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIdeltaB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIdeltaB but also a cytoplasmic isoform, CaMKIIdeltaC. In the present study, we demonstrate that expression of the deltaC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic deltaC isoform of CaMKII can lead to phosphorylation of Ca2+ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the deltaC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2+ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIdeltaC can mediate phosphorylation of Ca2+ regulatory proteins in vivo and provide evidence for the involvement of CaMKIIdeltaC activation in the pathogenesis of dilated cardiomyopathy and heart failure.