Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub-G1 fraction, annexin V staining, low DeltaPsi(m), and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro-apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This finding may help to identify the specific factors associated with the pro-apoptotic activities of ANT isoforms.