We determined the mitochondrial membrane status, presence of reactive oxygen species (ROS), and oxidative DNA adduct formation in normal human oral keratinocytes (NHOK) during senescence. The senescent cells showed accumulation of intracellular ROS and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), a major oxidative DNA adduct. Exposure of cells to H2O2 induced 8-oxo-dG accumulation in cellular DNA, which was rapidly removed in replicating NHOK. However, the 8-oxo-dG removal activity was almost completely abolished in the senescing culture. Both replicating and senescing NHOK expressed readily detectable 8-oxo-dG DNA glycosylase (hOGG1), the enzyme responsible for glycosidic cleavage of 8-oxo-dG. After exposure to H2O2, however, the intranuclear level of the hOGG1-alpha isoform was decreased in senescing but not in replicating NHOK. These results indicated that senescing NHOK accumulated oxidative DNA lesions in part due to increased level of endogenous ROS and impaired intranuclear translocation of hOGG1 enzyme upon exposure to oxidative stress.