The Na⁺/H⁺ exchanger isoform 1 (NHE1) and the Ca²⁺-independent phospholipase A2 VI (PLA2-VI) are pH sensitive proteins which exert both protective and detrimental effects in cardiac ischemia/reperfusion. In vivo, cardiac ischemia is associated with a reduction of intra- (pH_i) and extracellular pH (pH_o), as a consequence of inadequate blood flow and increased glycolytic metabolism. Here, we investigated the role of pH_o on injuries of HL-1 mouse cardiomyocytes after simulated ischemia (SI, 5 h 0.5% O₂ at pH_o 6.0 and 7.4) and reperfusion (SI/R, 4 or 8 h 21% O₂ at pH_o 7.4), and the regulation and function of PLA2-VI and NHE1 under these conditions. In accordance with previous studies, SI/R elicited mitochondrial fragmentation, cytochrome c release and caspase-3 activation, cytoskeletal disruption and necrotic cell death. Mitochondrial fragmentation, cytochrome c release and caspase-3 activation were attenuated after acidic compared to neutral SI. Inhibition of NHE1 by 5'N-ethylisopropylamiloride (EIPA, 5 mM), had no significant effect on necrosis, while inhibition of PLA2-VI activity by bromoenol lactone (BEL, 10 mM) elicited necrosis during normoxia and to a lesser extent after acidic SI, yet not neutral SI. Moreover BEL-treatment disrupted mitochondrial integrity. EIPA slightly reduced stress fibre content whereas BEL disrupted F-actin and cortactin integrity. NHE1 and the PLA2-VI isoform PLA2-VIA mRNA levels were reduced during SI. The NHE1 mRNA level was increased significantly above that in normoxic controls after reperfusion following acidic, yet not neutral SI. NHE1 protein level was downregulated during SI while PLA2-VIA protein level was unaffected during SI and SI/R. Regarding localization NHE1 localized to the plasma membrane during normoxia. This was disrupted by SI at both pH_o values, and partially recovered during reperfusion. A partial perinuclear translocation of PLA2-VIA was seen after neutral yet not after acidic SI/R. In conclusion, the mode of cell death and NHE1 and PLA2-VI expression patterns were dependent on pH_o during SI. Moreover PLA2-VI exerted net pro-survival effects under normoxia and in acidic SI and SI/R, in part by counteracting necrotic cell death. This effect of PLA2-VI was less pronounced in neutral SI and SI/R. NHE1 activity did not contribute to SI/R induced necrosis. Thus, the mode of cell death, and the roles and regulation of NHE1 and PLA2-VI, are at least in part determined by pH_o during SI. In addition to having clinically relevant implications, these findings can in part explain contradictory results obtained from previous studies of PLA2-VIA and NHE1 during cardiac ischemia/reperfusion. Necrosis-independent roles of NHE1 in control of death/survival balance and the background for the loss of the protective/the nature of the detrimental PLA2-VI activity apparently induced by neutral SI are under investigation.