Hyperbaric oxygen (HBO) therapy is successfully used for the treatment of a variety of conditions. However, exposure to high concentrations of oxygen is known to induce damage to cells, possibly due to an increased oxygen radical production. As reactive oxygen species also cause DNA damage, we investigated the DNA-damaging effect of HBO with the alkaline version of the single cell gel test (comet assay). Oxidative DNA base modifications were determined by converting oxidized DNA bases to strand breaks using bacterial formamidopyrimidine-DNA glycosylase (FPG), a DNA repair enzyme, which specifically nicks DNA at sites of 8-oxo-guanines and formamidopyrimidines. HBO treatment under therapeutic conditions clearly and reproducibly induced DNA damage in leukocytes of all test subjects investigated. Increased DNA damage was found immediately at the end of the treatment, while 24 h later, no effect was found. Using FPG protein we detected significant oxidative base damage after HBO treatment. DNA damage was detected only after the first treatment and not after further treatments under the same conditions, indicating an increase in antioxidant defences. DNA damage did not occur when the HBO treatment was started with a reduced treatment time which was then increased stepwise.