Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time. Reactive internucleotide groups were introduced in dsDNA immediately 5' or 3' to the oxidative lesion and one or two nucleotides 5' or 3' away from it. We showed that the Fpg protein specifically binds to the modified DNA duplexes. The binding efficiency varied with the position of the reactive group and was higher for the duplexes containing substituted pyrophosphate groups at the ends of pentanucleotide with the oxoG in the center. The nicking efficiency of the DNA duplexes containing the reactive groups one or two nucleotides 5' away from the lesion was higher as compared to non-modified DNA duplex bearing only the oxidative damage. We found two novel non-hydrolizable substrate analogs for the Fpg protein containing pyrophosphate and substituted pyrophosphate groups 3' adjacent to the oxoG. Using crosslinking, we revealed the phosphate groups, 3' and 5' adjacent to the lesion, which have specific contacts with nucleophilic amino acids from the E. coli Fpg protein active center. The crosslinking efficiency achieved 30%. The approaches developed can be employed in the studies of pro- and eucaryotic homologs of the E. coli Fpg protein as well as other repair enzymes.