Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil.