Iodinated contrast media (CM) are among the most frequently used drugs for intravenous administration in medical imaging. The study was performed to investigate cytogenetic effects in vitro of two contrast media (CM), Urografina 292 (meglumine amidotrizoate and sodium-ionic dimmer) and Reliev 60% (meglumine diatrizoate-ionic monomer) in cultures of human peripheral blood cells using the comet assay. In vitro experiments with CM were performed with peripheral blood cells of seven healthy individuals. Blood samples were collected in heparin-containing vials. Approximately 50 µL of cells were treated with 100 mg/mL of both CM and then incubated in an atmosphere supplemented with 5% CO₂ at 37ºC for 24 hours. Next, 10 µL of blood cells were collected after 4 and 24 hours for the comet assay. Doxorubicin was used as positive control. The genotoxic effect of both ionic CM caused increased DNA damage in both parameters of the comet assay (damage index and damage frequency) when compared to negative control group after 4 (P < 0.01) and 24 hours (P < 0.05) of exposure. Urografina 292 and Reliev 60% induced a considerable number of DNA breaks. Our results suggest that the tested CM possibly impaired the DNA repair system, since the damage observed 4 hours after exposure was also observed after 24-hour exposure.