Human alkyladenine DNA glycosylase (AAG) protects against alkylative and oxidative DNA damage, flipping damaged nucleotides out of double-stranded DNA and catalyzing the hydrolytic cleavage of the N-glycosidic bond to release the damaged nucleobase. The crystal structure of AAG bound to a DNA substrate reveals features of the active site that could discriminate between oxidatively damaged or alkylated purines and normal purines. A water molecule bound in the active site adjacent to the anomeric carbon of the N-glycosidic bond is suggestive of direct attack by water, with Glu125 acting as a general base. However, biochemical evidence for this proposed mechanism has been lacking. The structure also fails to explain why smaller pyrimidine nucleosides are excluded as substrates from this relatively permissive active site that catalyzes the excision of a structurally diverse group of damaged purine bases. We have used pH dependencies, site-directed mutagenesis, and a variety of substrates to investigate the catalytic mechanism of AAG. Single-turnover excision of hypoxanthine and 1,N(6)-ethenoadenine follows bell-shaped pH-rate profiles, indicating that AAG-catalyzed excision of these neutral lesions requires the action of both a general acid and a general base. In contrast, the pH-rate profile for the reaction of 7-methylguanine, a positively charged substrate, shows only a single ionization corresponding to a general base. These results suggest that AAG activates neutral lesions by protonation of the nucleobase leaving group. Glu125 must be deprotonated in the active form of the enzyme, consistent with a role as a general base that activates and positions a water nucleophile. Acid-base catalysis can account for much of the 10(8)-fold rate enhancement that is achieved by AAG in the excision of hypoxanthine. The prominent role of nucleobase protonation in catalysis by AAG provides a rationale for its specialization toward damaged purines while effectively excluding pyrimidines.