Objectives: Postconditioning (PC) the heart by brief repetitive cycles of ischemia/reperfusion (I/R) at the onset of reperfusion, is widely studied as a clinically relevant way to improve patient prognosis. The mechanisms underlying cardioprotection by PC are incompletely characterized, but roles for acidic pH_i, reduced mitochondrial damage, and altered activity of protein kinases important for cell survival have been proposed [1]. Here we investigate the roles of pH_o and signalling via protein kinase B (PKB), the mitogen-activated protein kinases (MAPKs) p38, ERK1/2, and JNK, and glycogen synthase kinase 3b (GSK3b) in I/R damage in HL-1 cardiomyocytes. Methods: HL-1 cells were exposed to 5 h of simulated ischemia (SI, 0.5% O₂) followed by simulated reperfusion (SR) at pH_o 6.0 or 7.4, essentially as in [2]. Kinase activity and mitochondrial integrity were assessed by Western blotting and confocal imaging, respectively. Results: PKB was markedly activated at the end of SI, followed by return to basal levels within 1 min of acidic, and 15 min of neutral SR. ERK1/2 was activated after SI and early in SR, whereas p38 MAPK was activated in an apparently pH-insensitive manner during the first 30 min of SR but not during SI. In agreement with this, the ERK- and p38 MAPK effector Msk1 was also activated during SR. Marked JNK activation occurred within 15 min of neutral SR, and was several-fold reduced at acidic SR. GSK3b activity appeared unaffected by SR. Finally, cytochrome c release and cell death after I/R were reduced at pH_o 6.0 compared to pHo 7.4. Conclusions: Acidic pH_o during SR protects against I/R damage, in part by preserving mitochondrial integrity. Moreover, JNK activation is strongly reduced at acidic pH_o. The roles of JNK, p38 MAPK, and ERK in I/R induced death and acidic protection are currently under investigation. 1. A.Granfeldt et al. (2009). Cardiovasc.Res. 83, 234-246 2. A.D.Andersen et al. (2009). Am J Physiol Cell Physiol 296, C1227-C1242