Endonuclease III of Escherichia coli is normally involved in the repair of oxidative DNA damage. Here, we have investigated a possible role of EndoIII in the repair of alkylation damage because of its structural similarity to the alkylation repair enzyme 3-methyladenine DNA glycosylase II. It was found that overproduction of EndoIII partially relieved the alkylation sensitivity of alkA mutant cells. Site-directed mutagenesis to make the active site of EndoIII more similar to AlkA (K120W) had an adverse effect on the complementation and the mutant protein apparently inhibited repair by competing for the substrate without base release. These results suggest that EndoIII might replace AlkA in some aspect of alkylation repair, although high expression levels are needed to produce this effect.