In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.