The cardiac sarco(endo)plasmic reticulum Ca²⁺-ATPase gene (ATP2A2) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a^−/− mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a^−/− mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a^−/− mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a ≈20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a^−/− animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved β-adrenergic response. Ca²⁺ uptake levels in SERCA2a^−/− cardiac homogenates were reduced by ≈50%. In isolated cells, relaxation and Ca²⁺ removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.