The recently devised domain peptide probe technique was used to identify and characterize critical domains of the cardiac ryanodine receptor (RyR2). A synthetic peptide corresponding to the Gly²⁴⁶⁰-Pro²⁴⁹⁵ domain of the RyR2, designated DPc10, enhanced the ryanodine binding activity and increased the sensitivity of the RyR2 to activating Ca²⁺: the effects that resemble the typical phenotypes of cardiac diseases. A single Arg-to-Ser mutation made in DPc10, mimicking the recently reported Arg²⁴⁷⁴-to-Ser²⁴⁷⁴ human mutation, abolished all of these effects that would have been produced by DPc10. On the basis of the principle of the domain peptide probe approach (see Model 1), these results indicate that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. This domain peptide approach opens the new possibility in the studies of the regulatory and pathogenic mechanisms of the cardiac Ca²⁺ channel.