The G/T mismatch specific thymine DNA glycosylase (EC 3.2.2.-) removes the mismatched T with opposite a G residue by its DNA N-glycosylase activity. It is one of the essential components of a functional DNA demethylation complex in chicken embryos, and which also contains 5-methylcytosine DNA glycosylase (5-MCDG), RNA helicase, and G/T mismatch DNA glycosylase activities. The chicken homologue of human G/T mismatch DNA glycosylase shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C- and N-terminal parts have about 50% identity. The recombinant chicken protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. Hemimethylated DNA is a better substrate than symmetrically methylated CpGs for the 5-MCDG activity. Deletion mutations indicate that G/T mismatch and 5-MCDG activities require the same areas of the N- and C-terminal parts of the protein.