Methylglyoxal is a direct-acting mutagen in Salmonella typhimurium TA100 and its mutagenicity is markedly enhanced in the presence of hydrogen peroxide. In addition, a mixture of methylglyoxal and hydrogen peroxide reacts with 2 -deoxyguanosine to form N 2 -acetyl-2 -deoxyguanosine. We examined whether the guanine residues in DNA were acetylated by methylglyoxal in the presence of hydrogen peroxide using the 3 2 P-postlabeling method. First,N 2 -acetyl-2 -deoxyguanosine 3 -monophosphate and N 2 -acetyl-2 -deoxyguanosine 3 ,5 -dephosphate were chemically synthesized as standard compounds for the analysis. Then calf thumus DNA (3.24 μmol) was treated with methylglyoxal (64.8 μmol) at pH 7.4 for 3 h at 37°C, and subsequently with hydrogen peroxide (64.8 μmol) at 37°C for 2 h. The adduct formation was analyzed using HPLC in combination with the 3 2 P-postlabeling method under the standard c conditions. N 2 -Acetyl-2 -deoxyguanosine was detected at levels of 2 6 nucleotides in double-stranded DNA and 1 5 nucleotides in single-stranded DNA. The estimated limit of detection by our method was 3 per 10 8 nucleotides.