Calcium (Ca2+) is a critical second messenger in cell signaling. Elevated intracellular Ca2+ can activate numerous Ca2+-regulated enzymes. These enzymes have different subcellular localizations and may respond to distinct modes of Ca2+ mobilization. In cardiac muscle, Ca2+ plays a central role in regulating contractility, gene expression, hypertrophy, and apoptosis. Many cellular responses to Ca2+ signals are mediated by Ca2+/calmodulin-dependent enzymes, among which is the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Putative substrates for CaMKII include proteins involved in regulating Ca2+ storage and release, transcription factors, and ion channels. The major isoform of CaMKII in the heart is CaMKIIδ. Two cardiac splice variants, CaMKIIδB and δC, differ in whether they contain a nuclear localization sequence. Our laboratory has examined the hypothesis that the nuclear δB and the cytoplasmic δC isoforms respond to different Ca2+ stimuli and have distinct effects on hypertrophic cardiac growth and Ca2+ handling. We have shown that pressure overload-induced hypertrophy differentially affects the nuclear δB and the cytoplasmic δC isoforms of CaMKII. Additionally, using isolated myocytes and transgenic mouse models, we demonstrated that the nuclear CaMKIIδB isoform plays a key role in cardiac gene expression associated with cardiac hypertrophy. The cytoplasmic CaMKIIδC isoform phosphorylates substrates involved in Ca2+ handling. Dysregulation of intracellular Ca2+ and resulting changes in excitation-contraction coupling characterize heart failure and can be induced by in vivo overexpression of CaMKIIδC and phosphorylation of its substrates. The differential location of CaMKII isoforms and their relative activation by physiological vs. pathological stimuli may provide a paradigm for exploring and elucidating how Ca2+/CaMKII pathways can serve as both friends and foes in the heart.