Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction and regulation of mitochondrial ROS production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating age-related alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats a decline in mitochondrial biogenesis occurs with aging. In aged vessels expression of the mitochondrial biogenesis factors (including Tfam, PGC1) was decreased. The vascular expression of complex I, III and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome C oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and down-regulation of COX may contribute to increased mitochondrial oxidative stress in aged endothelial cells.