The key role of mitochondria in the apoptotic process is well understood, but not many data are available regarding the specific role of mitochondrial DNA mutations in determining cell fate. We investigated whether two mitochondrial DNA mutations (L217R and L156R) associated with maternally-inherited Leigh syndrome may play a specific role in triggering the apoptotic cascade. Considering that different nuclear genetic factors may influence the expression of mtDNA mutations, we used a 143BTK(-) osteosarcoma cell line deprived from its own mtDNA in order to insert mutated mtDNAs. Analysis of mitochondrial features in these cybrids indicated that both mitochondrial DNA mutations produced evidence of biochemical, functional and ultrastructural modifications of mitochondria, and that these modifications were associated with an increased apoptotic proneness. Cybrids were highly susceptible to two different apoptotic stimuli, tumour necrosis factor-alpha and Staurosporin. The mechanism involved was the mitochondrial 'intrinsic' pathway, i.e. the caspase 9-driven cascade. More importantly, our results also indicated that the polarization state of the mitochondrial membrane, i.e. a constitutive hyperpolarization detected in cybrid clones, played a specific role. Interestingly, the different effects of the two mutations in terms of susceptibility to apoptosis probably reflect the deeper bioenergetic defect associated with the L217R mutation. This work provides the first evidence that hyperpolarization of mitochondria may be a 'risk factor' for cells with a deep ATPase dysfunction, such as cells from patients with maternally-inherited Leigh syndrome.