Compounds that contain chromate (Cr(VI)) are well-known carcinogens that are present in both industrial settings and the environment. The mechanism of carcinogenesis associated with these compounds is not well understood. This study focused on Cr(VI)-induced cell cycle arrest in human colon adenocarcinoma DLD1 cells. Treatment of the cells with Cr(VI) at 2.5 µM caused a growth arrest at the S phase. An increase in Cr(VI) concentration enhanced the growth arrest. Superoxide dismutase did not alter the Cr(VI)-induced S phase arrest. Catalase inhibited S-cell growth, indicating that H(2)O(2) is an important mediator in Cr(VI)-induced S phase arrest. Electron spin resonance spin-trapping measurements showed that incubation of cells with Cr(VI) generated hydroxyl radical (·OH). Catalase inhibited ·OH generation, indicating that H(2)O(2) was generated from cells stimulated by Cr(VI) and that H(2)O(2) functioned as a precursor of ·OH radical generation. p53, an oxidative response transcription factor, was activated upon Cr(VI) stimulation. Inhibition of p53 by introducing small hairpin RNA decreased S phase arrest induced by Cr(VI). These results support the following conclusions: (1) reactive oxygen species (ROS) are generated in Cr(VI)-stimulated DLD1 cells; (2) among the ROS generated, H(2)O(2) played a major role in causing S phase arrest in DLD1 cells; and (3) ROS mediated S phase arrest through a p53-dependent pathway.