Cellular senescence-elevated oxidative stress plays a critical role in age-associated vascular endothelial dysfunction. We investigated whether deficiency of mitochondrial cytochrome c oxidase (complex IV) is causally linked to increased oxidant generation during cellular aging using senescent (passage 45) and young (passage 3) pulmonary artery endothelial cells (PAEC). In senescent PAEC, levels of O2- and H2O2 were elevated onefold, respectively, compared to those in young cells. Lipid peroxidation and protein carbonyl contents in aged cells were increased more than twofold compared to young cells. To determine whether lack of complex IV in senescent cells contributed to the increased oxidant generation, complex IV activity in young cells was specifically inhibited using antisense oligonucleotides directed against the mRNA of complex IV subunits. Levels of O2- and H2O2 in PAEC treated with antisense oligonucleotides were elevated onefold, respectively, which correlated with a similar increase in lipid (110%) and protein (20%) oxidation, compared to control oligonucleotides-transfected cells. Moreover, levels of nitrosylated proteins in antisense-transfected cells were increased 30%, compared to controls. These data demonstrate that deficiency of complex IV in senescent cells enhances oxidative and nitrosative stress, which may be responsible for senescence-induced endothelial cell loss and dysfunction.