A multidisciplinary strategy was used to identify the molecular defect in a family with Leigh syndrome (LS). The propositus presented severe developmental delay, an ataxic-spastic gait and seizures. She died at 3.5 y of age from cardiorespiratory arrest. Postmortem examination disclosed pathological features typical of LS. A 12-y-old sister is affected with the same disease. Respiratory chain enzyme complex activities in skeletal muscle biopsy were normal. Adenosine triphosphate (ATP) synthesis during oxidative phosphorylation in skin fibroblasts mitochondria showed a severely hampered ATP production. Mitochondrial DNA sequencing revealed a new mutation in the ATPase 6 gene (T9176G). Site-directed mutagenesis in Escherichia coli strains was used to measure H+ pumping and ATP synthesis. Results were comparable to findings obtained in human cells. These data corroborate the use of E. coli strains as a feasible "animal" model for functional studies in pathogenic mutations of the ATPase 6 gene.