Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial β-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid β-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH NAD + ratio: the cells showed a highly elevated lactate pyruvate ratio in the suspending medium when incubated with glucose. The β-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the β-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid β-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of β-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH NAD + -redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal β-oxidation system.