Alpha B-crystallin (BC) is a small heat shock protein expressed at high levels in the myocardium where it protects from ischemia/reperfusion damage. Ischemia/reperfusion activates p38 MAP kinase, leading to the phosphorylation of BC on serine-59 (P- BC-S59), enhancing its ability to protect myocardial cells from damage. In the heart, ischemia/reperfusion also causes the translocation of BC from the cytosol to other cellular locations, one of which was recently shown to be mitochondria. However, it is not known whether BC translocates to mitochondria during ischemia and/or reperfusion, nor is it known whether BC phosphorylation takes place before or after translocation. In the present study, analyses of mitochondrial fractions isolated from mouse hearts subjected to various times of ex vivo ischemia/reperfusion showed that BC translocation to mitochondria was maximal after 20 min of ischemia, then declined steadily during reperfusion. Phosphorylation of mitochondrial BC was maximal after 30 min of ischemia, suggesting that at least in part, it occurred after BC association with mitochondria. Consistent with this was the finding that translocation of activated p38 to mitochondria was maximal after only 10 min of ischemia. The overexpression of BC-AAE, which mimics BC phosphorylated on serine-59, has been shown to stabilize mitochondrial membrane potential and to inhibit apoptosis. In the present study, infection of neonatal rat cardiac myocytes with adenovirus-encoded BC-AAE decreased peroxide-induced mitochondrial cytochrome c release. These results suggest that during ischemia, BC translocates to mitochondria, where it is phosphorylated and contributes to modulating mitochondrial damage upon reperfusion.