Ca2+ release mediated by the ryanodine receptor (RyR) regulates many important cell functions including excitation-contraction (E-C) coupling in skeletal muscle, by which membrane depolarization controls the opening of RyR via the dihydropyridine receptor. Among the three RyR subtypes, RyR-1 mediates skeletal muscle E-C coupling, whereas RyR-2 and RyR-3 cannot substitute for RyR-1. We carried out expression experiments using cultured mutant skeletal myocytes not having intrinsic intracellular Ca2+ release channels to study the structure-function relationship of amino acid residues 1303-1406 in RyR-1 (D2 region). In this region the amino acid sequences are highly divergent between RyR-1 and RyR-2, and the corresponding sequence is lacking in RyR-3. Expression of RyR-1 but not of RyR-2 rescued E-C coupling in the mutant cells. Deletion of either the entire D2 region or its N-terminal half from RyR-1 preserved the function of RyR-1 as a Ca2+ release channel but resulted in the loss of E-C coupling. Substitution of the D2 region for the corresponding sequence of RyR-2 had no effect on the function of RyR-1. These results indicate that the presence of the D2 region is critical for E-C coupling in skeletal muscle, although the D2 region alone cannot determine the functional difference between RyR-1 and RyR-2.