Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (Δ-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases : Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of Δ-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of Δ-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of Δ-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (@gR0) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0−86 %Δ-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating Δ-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50−55 %Δ-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of Δ-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of Δ-mtDNA in the target tissues of patients with mtDNA diseases.