Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H(2)O(2). However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase Cepsilon (PKCepsilon)-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCepsilon.