AIMS: To determine if mild hyperoxia induces senescence in retinal pigment epithelium (RPE) cells in vitro. METHODS: RPE340 cells and WI38 cells were grown in 20% oxygen and 40% oxygen until proliferative exhaustion. A combined senescence associated beta galactosidase (SABG) and 5-bromo-2'-deoxyuridine (BrdU) double labelling technique was performed at different times and labelled cells were counted. RESULTS: Cells grown in 40% oxygen stopped proliferating at an earlier population doubling level than when grown in 20% oxygen. An increase in SABG positive cells and decrease of BrdU positive cells in 40% oxygen developed at an earlier time than when grown in 20% oxygen. CONCLUSION: Mild hyperoxia induces premature senescence in a specific RPE cell line.