Hydrogen sulfide (H2S) produced by commensal sulfate-reducing bacteria, which are often members of normal colonic microbiota, represents an environmental insult to the intestinal epithelium potentially contributing to chronic intestinal disorders that are dependent on gene-environment interactions. For example, epidemiologic studies reveal either persistent sulfate-reducing bacteria colonization or H2S in the gut or feces of patients suffering from ulcerative colitis and colorectal cancer. However, a mechanistic model that explains the connection between H2S and ulcerative colitis or colorectal cancer development has not been completely formulated. In this study, we examined the chronic cytotoxicity of sulfide using a microplate assay and genotoxicity using the single-cell gel electrophoresis (SCGE; comet assay) in Chinese hamster ovary (CHO) and HT29-Cl.16E cells. Sulfide showed chronic cytotoxicity in CHO cells with a %C1/2 of 368.57 micromol/L. Sulfide was not genotoxic in the standard SCGE assay. However, in a modified SCGE assay in which DNA repair was inhibited, a marked genotoxic effect was observed. A sulfide concentration as low as 250 micromol/L (similar to that found in human colon) caused significant genomic DNA damage. The HT29-Cl.16E colonocyte cell line also exhibited increased genomic DNA damage as a function of Na2S concentration when DNA repair was inhibited, although these cells were less sensitive to sulfide than CHO cells. These data indicate that given a predisposing genetic background that compromises DNA repair, H2S may lead to genomic instability or the cumulative mutations found in adenomatous polyps leading to colorectal cancer.