It has been proposed that cellular ageing may be caused by loss of mitochondrial function due to the action of free radicals. To investigate this hypothesis, antigenic structures of the mitochondrial inner membrane/matrix and of the outer mitochondrial membrane of human diploid fibroblasts were monitored by immunoblotting at four stages during cellular lifespan in vitro. At the same time, specific activities of the enzymes oligomycin-sensitive ATPase (O-S ATPase), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) were assayed to assess the functional capacity of cellular oxidative phosphorylation and of the tricarboxylic acid cycle. No changes were found with ageing in inner mitochondrial membrane-associated matrix components, or in the activities of O-S ATPase and MDH. However GDH activity increased significantly with ageing in vitro, possibly indicating greater amino acid utilization for energy production in older cells. There was loss of an outer mitochondrial membrane antigen, of approximate molecular weight 60 kilodaltons (kDa), in the oldest cells tested, which may influence outer membrane transport capacity late in the cellular lifespan. Overall, the results fail to provide support for the hypothesis that ageing primarily results from free radical-induced impairment of mitochondrial function.