Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), a critical transducer of Ca²⁺ signaling, is a multifunctional protein kinase which can phosphorylate a wide range of substrates and regulate numerous cellular functions. The δ isoforms of CaMKII predominate in the heart and two splice variants of CaMKIIδ, δ_B and δ_C, have been demonstrated to be present in the adult mammalian myocardium. The δ_B isoform contains a nuclear localization signal (NLS) that is absent from δ_C, and consequently, the two isoforms have different subcellular localization. Recent work from our laboratory and others has implicated CaMKII in the development of cardiac hypertrophy and heart failure. The specific roles of these CaMKII isoforms in regulating cardiac function appear to be determined by their subcellular localization. The nuclear δ_B isoform plays a key role in hypertrophic gene expression, whereas the cytoplasmic δ_C isoform can affect excitation–contraction (E–C) coupling through phosphorylation of Ca²⁺ regulatory proteins and may also transduce signals leading to apoptosis. In addition, the nuclear δ_B and the cytoplasmic δ_C isoforms of CaMKII are differentially regulated in pressure overload-induced cardiac hypertrophy. This review focuses on evidence that CaMKII plays an essential role in transcriptional activation associated with cardiac hypertrophy, as well as the aberrant Ca²⁺ handling and apoptosis that may contribute to heart failure. The hypothesis that CaMKII isoform selective activation, localization and substrate phosphorylation lead to specificity in the resultant signaling pathways is discussed.