Four synthetic oligodeoxyribonucleotides of the sequence 5'-CCG1TG2G3G4ATATGGGCTG-3' were constructed with a 1',2'-[3H]deoxyguanosine located at one of the four sites indicated (1, 2, 3, or 4). This sequence was derived from a region of the Escherichia coli xanthine-guanine phosphoribosyltransferase gene where position 4 is a site frequently mutated by N-methyl-N'-nitrosourea as compared to sites 1-3. These four oligomers were alkylated in both single- and double-stranded form with N-methyl-N'-nitrosourea, and the relative amount of O6-methyldeoxyguanosine (O6-MedGuo) formed at each position was quantitated. Up to a 5-6-fold greater formation of O6-MedGuo was observed at positions 3 and 4 as compared to positions 1 and 2. This uneven distribution was only observed in oligomers in the double-stranded form, suggesting that secondary structure was an important determinant in generating the uneven distribution of O6-MedGuo. Comparisons between the extent of O6-MedGuo formation and mutation frequency at the four positions suggest that a difference in the formation of promutagenic adducts at specific sites is just one of the factors involved in the generation of mutagenic "hotspots." The novel method developed was applied to the study of formation of O6-MedGuo at specific sites; however, it should be suitable for studying the formation and repair of DNA adducts generated by a variety of chemicals in a wide variety of DNA sequences.