Mitochondria were prepared from three lymphoblast cell lines from patients with high percentage copy numbers of the human mtDNA 8993 mutation and compared to those prepared from related and non-related control cell lines. Rates of ATP synthesis with pyruvate/malate, succinate/rotenone, ascorbate/N'N'N'N' tetramethyl phenylene diamine were reduced to 67%, 58% and 54% of the control rates, respectively. The backward reaction measured as oligomycin sensitive ATPase was reduced to an average of 42% of that in controls. This mutation which changes a conserved leucine to an arginine in the putative membrane proton channel of mitochondrial ATPase effectively reduces the overall rate of oxidative phosphorylation.