Germ-free rats were fed purified diets containing sucrose (0.3%, wt/wt) as control or the synthetic disaccharide lactulose (0.3% wt/wt) and were then dosed orally with a human fecal suspension. After five weeks on the diets, these "human flora-associated" rats were transferred to diets in which the sucrose or lactulose level was raised to 3% (wt/wt). Four weeks later, the rats from each dietary group were dosed orally with saline (controls) or 1,2-dimethylhydrazine dihydrochloride (DMH, 15 mg/kg). Sixteen hours after the carcinogen dose, the rats were killed, colon cells were isolated, and the degree of DNA damage in the cells was assessed using the single cell microgel electrophoresis (Comet) assay. Samples of cecal contents were also removed from the saline-treated rats to determine changes in composition of the gut microflora. No significant diet-related differences in cecal bacterial numbers were observed, although there was a trend for the numbers of lactobacilli to be higher in the lactulose-fed animals. DNA damage in colon cells was quantified by measurement of the length of the cellular DNA image after electrophoresis, and the evaluated cells were assigned to classes according to the degree of damage: undamaged (< 40 microns), moderately damaged (40-80 microns), or severely damaged (> 80 microns). There were no significant diet-related differences in DNA damage in colon cells from rats treated with saline. Treatment with DMH induced a highly significant increase in DNA damage in sucrose- and lactulose-fed rats. However, in the rats treated with DMH, the degree of DNA damage was significantly (p < 0.05) less in the lactulose-fed animals than in those given the sucrose diet, as evidenced by a decrease in the percentage of cells with severe damage. In the sucrose-fed rats, severely damaged cells accounted for 33% of the total compared with only 12.6% in the lactulose-fed DMH-treated animals. The results of this study, which was performed in human flora-associated rats to provide data of relevance to humans, indicate that lactulose consumption offered a degree of protection from the genotoxic effects in the colonic mucosa of a known colon carcinogen.