Retinal pigment epithelial cell dysfunction mediated by reactive oxygen intermediates has been suggested as a possible cause of age-related macular degeneration. To test the hypothesis that retinal pigment cells are susceptible to genetic damage mediated by reactive oxygen intermediates, retinal pigment epithelial cells were treated with 50 μM-200 μM of hydrogen peroxide in vitro. Damage to mitochondrial DNA and three nuclear loci were assessed using quantitative polymerase chain reaction. Hydrogen peroxide treatment of retinal pigment epithelial cells resulted in significantly increased mitochondrial DNA damage. Significant mitochondrial DNA damage occurred rapidly and was not completely repaired within 3 hr post-treatment. By contrast, no DNA damage was observed in three different nuclear loci (β- globin gene cluster, hprt, and β-polymerase genes). Hydrogen peroxide treatment of retinal pigment epithelial cells also resulted in decreased mitochondrial redox function compared to controls, consistent with increased mitochondrial DNA damage. Consequently, retinal pigment epithelial cell mitochondrial DNA appears susceptible to hydrogen peroxide mediated damage in vitro, and thus, may serve as a catalyst in the initial events leading to retinal pigment epithelial cell dysfunction in vivo.