Evidence obtained in the last two decades indicates that calsequestrin (CSQ2), as the major Ca(2+)-binding protein in the sarcoplasmic reticulum of cardiac myocytes, communicates changes in the luminal Ca(2+) concentration to the cardiac ryanodine receptor (RYR2) channel. This review summarizes the major aspects in the interaction between CSQ2 and the RYR2 channel. The single channel properties of RYR2 channels, discussed here in the context of structural changes in CSQ2 after Ca(2+) binding, are particularly important. We focus on five important questions concerning: (1) the method for reliable detection of CSQ2 on the reconstituted RYR2 channel complex; (2) the power of the procedure to strip CSQ2 from the RYR2 channel complex; (3) structural changes in CSQ2 upon binding of Ca(2+) which cause CSQ2 dissociation; (4) the potential role of CSQ2-independent regulation of the RYR2 activity by luminal Ca(2+); and (5) the vizualization of CSQ2 dissociation from the RYR2 channel complex on the single channel level. We discuss the potential sources of the conflicting experimental results which may aid detailed understanding of the CSQ2 regulatory role. Although we mainly focus on the cardiac isoform of the proteins, some aspects of more extensive work carried out on the skeletal isoform are also discussed.