The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U.G mismatches (Gallinari, P., and Jiricny, J. (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug). It has also been described to remove 3,N(4)-ethenocytosine (epsilonC) from epsilonC.G mismatches (Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8508-8513). We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E. coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U.G and T.G mismatches, respectively, we conclude that Mug does not repair U.G or T.G mismatches in vivo. A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E. coli. Cell-free extracts from mug(+) ung cells show very little ability to remove uracil from DNA, but can excise epsilonC. The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E. coli that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde.