The regulation of intracellular Ca2+ signaling is an important aspect of how anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins regulate cell death and cell survival. At the endoplasmic reticulum (ER) the Bcl-2 homology (BH) 4 domain of Bcl-2 is known to bind to and inhibit both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs). Besides this, drugs that target the hydrophobic cleft of Bcl-2 have been reported to deplete ER Ca2+ stores in an IP3R- and RyR-dependent way. This suggests that the hydrophobic cleft of Bcl-2 may also be involved in regulating these ER-located Ca2+-release channels. However, the contribution of the hydrophobic cleft on the binding and regulatory properties of Bcl-2 to either IP(3)Rs or RyRs has until now not been studied. Here, the importance of the hydrophobic cleft of Bcl-2 in binding to and inhibiting the RyR was assessed by using a genetic approach based on site-directed mutagenesis of Bcl-2's hydrophobic cleft and a pharmacological approach based on the selective Bcl-2 hydrophobic cleft inhibitor, ABT-199. Both binding assays and single-cell Ca2+ measurements indicated that RyR binding and the inhibition of RyR-mediated Ca2+ release by Bcl-2 is independent of its hydrophobic cleft. (C) 2015 Elsevier Inc. All rights reserved.