In mature cardiac myocytes, Ca²⁺ influx through the L-type Ca²⁺ channel activates the ryanodine receptor and triggers Ca²⁺ release from the sarcoplasmic reticulum (SR). This Ca²⁺ signal amplification, termed Ca²⁺-induced Ca²⁺ release (CICR), occurs within the junctional membrane complex between the plasma membrane and the SR, and is essential for cardiac excitation-contraction (E-C) coupling. On the other hand, Ca²⁺ available during E-C coupling is predominantly derived from Ca²⁺ influx in embryonic cardiac myocytes. To examine the role of the intracellular Ca²⁺ store in immature cardiac myocytes, we have generated knockout mice lacking the cardiac type of the ryanodine receptor (RyR-2), or junctophilin (JP-2) contributing to formation of the junctional membrane complex. Both RyR-2- and JP-2-knockout mice show lethality at early embryonic stages immediately after beginning of heart beating. The loss of RyR-2 produced abnormal SR elements exhibiting vacuolated structures and Ca²⁺-overloading in embryonic cardiac myocytes. In JP-2-deficient cardiac myocytes, formation of junctional membrane complexes, called pheripheral couplings, was disturbed, and abnormal Ca²⁺ transients without spatial and temporal synchronization were observed. Therefore, the knockout mice have demonstrated that RyR-2-mediated Ca²⁺ release at the junctional membrane complex is essential for cellular Ca²⁺ homeostasis in immature cardiac myocytes.