The BH3-only proteins, including BNip3, are members of the Bcl-2 family of cell death-regulating factors. Whereas proteins such as Bax and Bak play a central role in most forms of apoptosis, the BH3-only proteins appear to modulate apoptosis through cell type- and signal-specific pathways. This review will focus on our studies of the specific role of BNip3 in cardiac myocyte apoptotic signaling during ischemia. We recently showed that hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the [pH]_o drops below 6.5. A micro-array analysis of 20,000 genes identified the pro-apoptotic Bcl-2 family member BNip3 as one of the most strongly upregulated of >100 hypoxia-inducible genes in cardiac myocytes. BNip3 mRNA increased by 12-fold, and BNip3 protein by sixfold, during 24 h of hypoxia; BNip3 protein accumulation was further enhanced by acidosis. BNip3 was loosely bound to mitochondria under conditions of neutral hypoxia but became more tightly associated at acid pH, coincident with opening of the mitochondrial permeability transition pore (MPTP). Subsequent DNA fragmentation and cell death were not blocked by caspase inhibitors, but were inhibited by antisense BNip3 oligonucleotides and MPTP inhibitors, indicating that BNip3 activates an atypical programmed death pathway with features of both apoptosis and necrosis. The sequential induction and activation of BNip3 by hypoxia and acidosis provide a molecular basis for the observation that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.