DNA mismatch binding in vitro, resistance to DNA methylation damage, and spontaneous mutation rates were examined in human colorectal adenocarcinoma cell lines. Of 11 cell lines, 3 (DLD1, HCT15, and LoVo) were defective in mismatch binding. All three lines had a mutator phenotype. These properties indicate that DLD1 and HCT15 may, like LoVo, carry mutations in the mismatch recognition protein hMSH2. Mismatch binding was normal in the remaining eight lines, including HCT116 in which a second mismatch repair protein, hMLH1, is defective. Two lines, SW620 and SW48, did not express detectable levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. SW620 exhibited the expected sensitivity to N-methyl-N-nitrosourea. In contrast, SW48 cells were highly resistant to N-methyl-N-nitrosourea and also slightly to methyl methanesulfonate, indicating that they are tolerant to DNA methylation damage. SW48 exhibited the spontaneous mutator phenotype and microsatellite instability that are hallmarks of a defect in mismatch repair. This cell line provides evidence for the association between methylation tolerance and defective mismatch correction in human colorectal carcinoma cells. The properties of methylation-tolerant, mismatch repair-defective cells identify possible selective pressures that might facilitate the natural selection of mismatch repair-defective tumors.