In earlier work, we showed that low dietary folate induced intestinal tumors in BALB/c mice. In this study, our goal was to examine the effect of the same diets on a strain that is more resistant to tumorigenesis (C57Bl/6). We also questioned whether supplementation of the folate-deficient diet (FD) with betaine, an alternate methyl donor, would influence tumor formation. C57Bl/6 mice were fed the same diets [control diet (CD) with 2 mg folate/kg diet and FD with 0.3 mg folate/kg diet] as those in our previous study for 1 y, but they did not develop tumors. We also fed BALB/c mice the FD or FD supplemented with betaine for 1 y, but there was no change in tumor incidence. To determine the relative contributions of DNA damage and altered methylation patterns, we measured intestinal dUTP:dTTP ratios, phosphorylated histone H2AX (p-H2AX) staining, and global DNA methylation in both strains. Only BALB/c mice showed changes due to diet in dUTP:dTTP (from 2.19 +/- 0.20 in CD to 2.77 +/- 0.18 in FD; P = 0.05) and in p-H2AX staining (from 14.10 +/- 3.59% in CD to 22.40 +/- 2.65% in FD; P = 0.054). In BALB/c mice only, FD tended to have less (P = 0.06) global DNA methylation than CD. Although the FD increased plasma homocysteine and the betaine-supplemented FD lowered plasma homocysteine, the latter diet did not reduce tumor incidence. We conclude that plasma homocysteine is not likely to be associated with tumorigenesis in our model. However, DNA damage plays a critical role in initiating tumorigenesis when dietary folate is low and methylation changes may also be contributory.