Organic nitrates act as endothelium-independent vasodilators of blood vessels. For these reasons, nitroglycerin (GTN) is one of the most widely used anti-ischemic drugs. The chronic efficacy of nitrates is blunted because of the development of nitrate tolerance and endothelial dysfunction. Recent data indicate that GTN induced reactive oxygen and nitrogen species (RONS) formation accounts for both phenomena. In 2005, Andreassi demonstrated that organic nitrate therapy increased DNA damage (Mol. Med. 2005). This report indicates our lack of understanding of molecular mechanism contributing to nitrate tolerance development. Following this initial trend, we conducted our experiments with the cell culture system using Ea.hy 926 cells. After GTN treatment we detected presence of oxidative DNA damage and DNA strand breaks in response to GTN. We proceeded with investigations on the animal model. After 3 days of continuous GTN treatment, as a correlate of clinical nitrate tolerance in patients, presence of the endothelial dysfunction and nitrate tolerance was confirmed. Additionally, we were able to detect increased cardiac RONS formation. With the help of immuno staining of the aortic tissue we found substantial elevation in the levels of 8-oxo-guanine and O6-methyl-guanine. Currently, it is still enigmatic whether noted increase in DNA damage during treatment with GTN is simply due to the accumulation of DNA damage under nitrate-induced oxidative stress or due to a more complex mechanisms such as inhibition of DNA repair machinery. In order to elucidate exact mechanism of action for GTN-induced DNA damage further investigations will be conducted on mice with impaired DNA repair machinery.