Pyridyloxobutylation of DNA yields adducts that react with O6-alkylguanine-DNA alkyl-transferase (AGT) to prevent the repair of O6-methylguanine (O6-mG). The chemical characterization of pyridyloxobutyl adducts has been confounded by their instability under DNA hydrolysis conditions. They decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) during the chemical or enzymatic hydrolysis of DNA. The goal of these studies was to determine which bases are pyridyloxobutylated to form AGT-reactive adducts. The model pyridyloxobutylating agent, 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc), was reacted with either poly(dAdT) or poly(dGdC) to generate DNA substrates for reaction with AGT. Only the pyridyloxobutylated poly(dGdC) was able to prevent the ability of partially purified rat liver AGT to repair O6-mG. These results paralleled those obtained for the corresponding methylated substrates. These studies are consistent with the pyridyloxobutylation of GC base pairs and not AT base pairs in the DNA to generate a substrate for AGT. In order to distinguish between the formation of reactive adducts at C residues versus G residues, two oligomers were designed that were complementary to one another. One oligomer contained A, T, and G residues, whereas its complement contained T, A, and C residues. Only the dG-containing oligomer reacted with NNKOAc to generate an AGT-reactive adduct, again paralleling the results obtained for a methylating agent. These results demonstrate that pyridyloxobutylation of only guanine residues produces adducts that react with AGT. These AGT-reactive guanine adducts are relatively stable within DNA, with a half-life of 1-2 weeks at 37 degrees C. They represent up to 70% of the total HPB-releasing adducts in the NNKOAc-treated DNA. We postulate that a potential AGT-reactive adduct is an O6-(pyridyloxobutyl)guanine adduct.