Deamination of cytosine residues contributes to the appearance of uracil in DNA. Uracil DNA glycosylase (UDG) initiates uracil excision repair to safeguard the genomic integrity. To study the mechanism of uracil excision in mycobacteria (organisms with G+C rich genomes), we have purified UDG from Mycobacterium smegmatis by more than 3000-fold. The molecular mass of M. smegmatis UDG, as determined by SDS/PAGE, is approximately 25 kDa and it shows maximum activity at pH 8.0. The N-terminal sequence analysis shows that the initiating amino acid, formyl-methionine is cleaved from the mature protein. More interestingly, unlike Escherichia coli UDG, which forms a physiologically irreversible complex with the inhibitor protein Ugi, M. smegmatis UDG forms a dissociable complex with it. M. smegmatis UDG excises uracil from the 5'-terminal position of the 5'-phosphorylated substrates. However, its excision from the 3'-penultimate position is extremely poor. Similar to E. coli UDG, M. smegmatis UDG also uses pd(UN)p as its minimal substrate. However, in contrast to E. coli UDG, which excises uracil from different loop positions of tetraloop hairpin substrates with highly variable efficiencies, M. smegmatis UDG excises the same uracil residues with comparable efficiencies. Kinetic parameters (Km and Vmax) for uracil release from synthetic substrates suggest that M. smegmatis UDG is an efficient enzyme and better suited for molecular biology applications. We discuss the usefulness of the distinct biochemical properties of M. smegmatis UDG in the possible design of selective inhibitors against it.