Myocardial ischemia-reperfusion (IR) injury occurs due to acute myocardial infarction (AMI) and triggers a variety of pathological processes that culminate in necrotic cell death. The protein kinase C (PKC) kinase family plays an important role in mediating the endogenous cardioprotective response to IR. Previously, our lab developed rationally designed PKC isozyme-selective regulatory compounds which were used to elucidate the role of specific PKC isozymes in mediating the protective response to myocardial IR (1). These studies found that selective activation of εPKC before ischemia (preconditioning) or immediately at the onset of reperfusion (postconditioning) can protect the heart from IR injury. The cardioprotective target of εPKC is thought to reside within mitochondria, as εPKC directly regulates opening of the mitochondrial permeability transition pore (mPTP) and mitoKATP channels (2). In order to access intramitochondrial targets, εPKC must first transverse the mitochondrial outer membrane, however, the mechanism for mitochondrial import of εPKC remains to be described. HSP90 is a member of the heat-shock-protein family of molecular chaperones and is activated by cellular stresses including IR injury. Recent evidence suggests that HSP90 also plays a role in mediating the mitochondrial import of cytosolic proteins, in tandem with TOM (translocase of the outer membrane) (3). The aim of the current study was to determine whether the HSP90-TOM import pathway plays a role in the mitochondrial import of εPKC. We employed an ex vivo rat heart model of myocardial IR in combination with electron microscopy (EM) and western blotting and found that IR induced significant mitochondrial import of εPKC. EM analysis showed that mitochondrial εPKC was predominantly localised on the inner mitochondrial membrane. To determine whether IR-induced mitochondrial import of εPKC required the HSP90-TOM import pathway, we used the HSP90-specific antagonist geladanamycin (GA). Infusion with 5μM GA at reperfusion prevented mitochondrial import of εPKC and dramatically worsened IR injury when compared to hearts exposed to IR alone. Biochemical analysis of mitochondrial εPKC revealed that εPKC forms a complex with both HSP90 and TOM20 at the mitochondria following exposure to IR but not under normoxic conditions. Co-precipitation of mitochondrial εPKC with HSP90 or TOM20 was prevented by GA at reperfusion. These findings demonstrate that cardiac IR induces the mitochondrial import of εPKC, which is mediated by the HSP90-TOM import pathway and is a required step in the endogenous cardioprotective response to IR. This is the first description of the mitochondrial import of εPKC and provides a novel mechanistic insight into εPKC-mediated cardioprotection.