Background Mitochondria are critical to cardiac injury during reperfusion as a result of damage sustained during ischemia, including the loss of bcl-2. We asked if bcl-2 depletion not only leads to selective permeation of the outer mitochondrial membrane (MOMP) favoring cytochrome c release and programmed cell death, but also favors opening of the mitochondrial permeability transition pore (MPTP). An increase in MPTP susceptibility would support a role for bcl-2 depletion mediated cell death in the calcium overload setting of early reperfusion via MPTP as well as later in reperfusion via MOMP as myocardial calcium content normalizes. Methods Calcium retention capacity (CRC) was used to reflect the sensitivity of the MPTP opening in isolated cardiac mitochondria. To study the relationship between bcl-2 inhibition and MPTP opening, mitochondria were incubated with a bcl-2 inhibitor (HA14-1) and CRC measured. The contribution of preserved bcl-2 content to MPTP opening following ischemia-reperfusion was explored using transgenic bcl-2 overexpressed mice. Results CRC was decreased in mitochondria following reperfusion compared to ischemia alone, indicating that reperfusion further sensitizes to MPTP opening. Incubation of ischemia-damaged mitochondria with increasing HA14-1concentrations increased calcium-stimulated MPTP opening, supporting that functional inhibition of bcl-2 during simulated reperfusion favors MPTP opening. Moreover, HA14-1 sensitivity was increased by ischemia compared to non-ischemic controls. Overexpression of bcl-2 attenuated MPTP opening in following ischemia-reperfusion. HA14-1 inhibition also increased the permeability of the outer membrane in the absence of exogenous calcium, indicating that bcl-2 inhibition favors MOMP when calcium is low. Conclusions The depletion and functional inhibition of bcl-2 contributes to cardiac injury by increasing susceptibility to MPTP opening in high calcium environments and MOMP in the absence of calcium overload. Thus, ischemia-damaged mitochondria with decreased bcl-2 content are susceptible to MPTP opening in early reperfusion and MOMP later in reperfusion when cytosolic calcium has normalized.