The induction of DNA damage by commonly used printing ink pigments, C.I. pigment orange 5 (C.I. 12075) and C.I. pigment yellow 12 (C.I. 21090), was investigated in freshly isolated rat hepatocytes with the comet assay. C.I. pigment yellow 12 is a 3,3´-dichlorobenzidine-based diarylide pigment, and C.I. pigment orange 5 is a naphthol-azo pigment. The pigments are virtually insoluble in aqueous solutions, and they have not been tested extensively for toxicological effects. C.I. pigment orange 5 increased the levels of DNA damage at 5 µg/ml (P < 0.02) and C.I. pigment yellow 12 at 20 µg/ml (P < 0.002). The effect of incubation time (20, 40, and 80 min) of the same concentrations of the pigments was tested. The levels of DNA damage were increased up to 80 min. Both pigments produced DNA damage that was in the same range as the food carcinogen 2-amino-3,8-dimethylimidazo[4,5f]quinoxaline. Our data indicate that both C.I. pigment orange 5 and C.I. pigment yellow 12 are genotoxic in hepatocytes with metabolizing capacities. However, further investigation of the metabolism and disposition are required for the evaluation of the safety of these pigments.