We have reported that mitochondrial DNA-depleted ρ⁰ cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ⁰ cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ⁰ cells have features simulating aged cells. SK-Hep1 hepatoma ρ⁰ cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ⁰ cells. These results support the mitochondrial theory of aging, and suggest that ρ⁰ cells could serve as an in vitro model for aged cells.