Members of the Bcl-2 protein family play a central role in the regulation of apoptosis. Some of their physiological effects are mediated by modulation of the ER localized inositol trisphosphate receptor Ca²⁺ release channel (InsP₃R). In previous studies we determined that Bcl-XL, an antiapoptotic Bcl-2 protein, bound to the InsP₃R to increase its sensitivity to InsP₃ and enhance spontaneous Ca²⁺ signalling (White et al., 2005; Li et al., 2007). The present study tested the hypothesis that the structurally related family members Bcl-2 and Mcl-1 had similar effects on InsP₃R-dependent Ca²⁺ handling. Bcl-2 or Mcl-1 were stably transfected into wild type DT40 cells endogenously expressing all three InsP₃R isoforms, or into DT40 cells genetically deficient in InsP₃Rs (DT40-InsP₃R-KO). The effect of Bcl-2 and Mcl-1 on ER store content and InsP₃R-dependent Ca²⁺ release was monitored directly in permeabilized cells loaded with the low affinity indicator mag-fura-2. In the DT40-WT background, the steady-state [Ca²⁺]_ER was significantly lower in Bcl-2 and Mcl-1 expressing cells; 30.3 ± 1 μM and 28.8 ± 1μM respectively (mean ± SEM), compared to vector only expressing controls 53.9 ± 3 μM (P < 0.001, ANOVA, n ≥ 36). The lowering of steady-state [Ca²⁺]_ER by Bcl-2 and Mcl-1 was partially reversed by application of heparin (100 μgml-1), an InsP₃R antagonist, during store loading. In addition, the filling state of the ER stores in DT40-InsP₃R-KO cells expressing Bcl-2 (55.6 ± 2 μM) and Mcl-1 (52.7 ± 2 μM) was not significantly different from DT40-InsP₃R-KO controls (59 ± 2 μM; n ≥ 69). These data suggest that InsP₃Rs are required for Bcl-2 and Mcl-1 to regulate store content. The effect of Bcl-2 and Mcl-1 on InsP₃-mediated Ca²⁺ release was determined by recording [Ca²⁺]_ER in response to 0.1 or 10 μM InsP₃ and examining the first order derivative of the release phase (d[Ca²⁺]_ER/dt) as a function of [Ca²⁺]_ER. Cells expressing Bcl-2 and Mcl-1 displayed faster Ca²⁺ release in response to 0.1 μM InsP₃ but not 10 μM. Thus, Bcl-2 and Mcl-1 increase the apparent sensitivity of InsP₃R-dependent Ca²⁺ release to low levels of InsP₃. Fura-2 imaging experiments were carried out to assess the effect of Bcl-2 and Mcl-1 expression on whole cell Ca²⁺ signalling. In the absence of stimulation, intact DT40-WT cells display spontaneous InsP₃R-dependent Ca²⁺ oscillations. In cells expressing empty vector the mean oscillation frequency was 3.45 ± 0.1 mHz; however, oscillation frequency was significantly higher in both Bcl-2 (6.18 ± 0.2 mHz) and Mcl-1 (8.19 ± 0.3) mHz expressing cells (P < 0.001, Mann-Whitney test, n ≥ 313). Taken together, these data demonstrate that Bcl-2 and Mcl-1 regulate [Ca²⁺]_ER handling and InsP₃R-dependent Ca²⁺ signalling in ways that are qualitatively similar to Bcl-XL.