We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5′-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation). The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites. All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A). The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide. The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.