Ceramide has been proposed to be a mediator of replicative senescence. Our aim was to determine whether ceramide induces senescence in vascular endothelial cells. Human umbilical vein endothelial cells were cultured to different population doubling levels and ceramide levels were quantitated. The endogenous levels of ceramide increased 2.4-fold with senescence onset. Low passage cells were chronically treated with exogenous C(6)-ceramide. This treatment induced a senescent phenotype as measured by an inhibition of cell proliferation and DNA replication while increasing senescence-associated beta-galactosidase expression. This is the second cell type in which ceramide induces senescence, thus implicating ceramide as a general mediator of cellular senescence.