The cytosolic small heat shock protein alphaB-crystallin (alphaBC) is a molecular chaperone expressed in large quantities in the heart, where it protects from stresses such as ischemia-reperfusion (I/R). Upon I/R, p38 MAP kinase activation leads to phosphorylation of alphaBC on Ser(59) (P-alphaBC-S59), which increases its protective ability. alphaBC confers protection, in part, by interacting with and affecting the functions of key components in stressed cells. We investigated the hypothesis that protection from I/R damage in the heart by P-alphaBC-S59 can be mediated by localization to mitochondria. We found that P-alphaBC-S59 localized to mitochondria isolated from untreated mouse hearts and that this localization increased more than threefold when the hearts were subjected to ex vivo I/R. Mitochondrial P-alphaBC-S59 decreased when hearts were treated with the p38 inhibitor SB-202190. Moreover, SB-202190-treated hearts exhibited more tissue damage and less functional recovery upon reperfusion than controls. I/R activates mitochondrial permeability transition (MPT) pore opening, which increases cell damage. We found that mitochondria incubated with a recombinant mutant form of alphaBC that mimics P-alphaBC-S59 exhibited decreased calcium-induced MPT pore opening. These results indicate that mitochondria may be among the key components in stressed cells with which P-alphaBC-S59 interacts and that this localization may protect the myocardium, in part, by modulating MPT pore opening and, thus, reducing I/R injury.