The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution. The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity. Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft. The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.