Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. CS cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from CS-A and CS-B patients present an altered redox balance with increased steady-state levels of intracellular ROS and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K-ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild-type genes. High resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS-A and CS-B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of CS patients and provide novel tools valuable for both diagnosis and therapy. (c) 2012 The Authors Aging Cell (c) 2012 Blackwell Publishing Ltd/Anatomical Society of GreatBritain and Ireland.