Iron accumulates as a function of age in many tissues and is associated with age-related pathologies. Although the molecular basis of this change is not known, it may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary (IMR90) and SV-40-transformed (SV-IMR90) human fibroblast cells were studied in culture as a model of cellular senescence. Total iron content increased exponentially in IMR90s during cellular senescence, whereas iron content in SV-IMR90s remained unchanged under identical conditions. Altering intracellular iron levels by administration of ferrous iron-citrate or subtoxic levels of iron chelators (desferrioxamine and salicyladehyde isonicotinoyl hydrazone) had no effect on cellular lifespan in either cell type. However, altering cellular senescence in IMR90s did change the kinetics of intracellular iron accumulation. Inducing early senescence in IMR90s with low-dose hydrogen peroxide concomitantly accelerated intracellular iron accumulation. Delaying senescence in IMR90s with the mitochondrial antioxidant N-tert-butyl-hydroxylamine (NtBHA) concomitantly attenuated intracellular iron accumulation. Furthermore, there was no change in intracellular iron content in SV-IMR90s when treated with either hydrogen peroxide and/or NtBHA. These data suggest that iron accumulation occurs as a consequence of cellular senescence in cultured human cells. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells.