We compared mitochondrial respiratory chain function, mitochondrial DNA (mtDNA) integrity, and oxidative stress levels in muscle, myoblasts, fibroblasts and cybrids, from 12 amyotrophic lateral sclerosis (ALS) patients with 28 control samples. Mitochondrial respiratory chain enzyme activities were normal in muscle, myoblast and fibroblast cultures from ALS patients, as were levels of mtDNA in muscle. Rearranged muscle mtDNA species were not detected by Southern blot hybridization in any of the samples and no difference was found in the number of deleted mtDNA species detected by long-range PCR. Platelet-derived cybrid studies confirmed the absence of a systemic mtDNA abnormality. Aconitase activity measurements did not indicate increased oxidative damage in muscle tissue, or in myoblasts or fibroblasts from ALS patients cultured under basal conditions. We did, however, find an increased sensitivity to oxidative stress in myoblasts from ALS patients exposed to paraquat. This altered sensitivity appears to be due to a nuclear rather than a mtDNA abnormality. Motor neurons have a large relative size and metabolic activity, and would be expected to be exposed to a greater degree of oxidative stress than most tissues throughout life. In addition, neurons are postmitotic cells, with poor regenerative potential. We do not have a ready method to study this in neural tissue of living patients, but the oxidative stress identified in myoblasts would translate into oxidative damage more readily in motor neurons than in other tissues.