The type 2 ryanodine receptor (RyR2) mediates sarcoplasmic reticulum (SR) calcium release which is critical for cardiac excitation-contraction coupling. RyR2 is composed of ~5000 amino acids with C-terminal transmembrane domains forming the channel pore while the bulk of the protein faces the cytoplasm. Cardiac myosin binding protein-C (cMyBP-C) is a modular protein associated with the sarcomere thick filament through its C-terminus interactions with myosin and titin. cMyBP-C is thought to regulate myocardial contractility by modifying the actin-myosin association primarily through its N-terminal region. We have isolated cMyBP-C as a putative RyR2-binding partner by yeast two-hybrid screening of a human cardiac cDNA library. The RyR2 interaction with cMyBP-C was also verified by co-immunoprecipitation assays following co-expression of both recombinant human polypeptides in mammalian HEK293 cells (n = 5), and importantly between the native proteins from pig cardiac muscle homogenates (n = 5). Further mapping studies were carried out using co-immunoprecipitation assays on a series of RyR2 and cMyBP-C truncated fragments co-expressed in HEK293 cells in order to identify the minimal interacting regions. Cumulative data (n ≥ 5) following densitometry analysis indicated robust binding between the RyR2 N-terminal (amino acids 161-759) and cMyBP-C C-terminal (amino acids 820-972) domains. Additional experiments demonstrated that the corresponding RyR1 and RyR3 N-terminal fragments are also capable of interaction with the C-terminus of cMyBP-C (n =6 for both constructs), suggesting that the RyR:MyBP-C association could be conserved in both skeletal and cardiac muscle. The above results suggest that the RyR2 interaction with cMyBP-C is likely to be physiologically relevant. This novel, potential modulation of RyR2-mediated SR Ca2+ release by a sarcomere component could provide a retrograde regulation mechanism for cardiac excitation-contraction coupling.