Escherichia coli possesses two different DNA repair glycosylases, Tag and AlkA, which have similar ability to remove the alkylation product 3-methyladenine from double-stranded DNA. In this study we show that these enzymes have quite different activities for the excision of 3-methyladenine from single-stranded DNA, AlkA being 10-20 times more efficient than Tag. We propose that AlkA and perhaps other glycosylases as well may have an important role in the excision of base damage from single-stranded regions transiently formed in DNA during transcription and replication.