DNA duplex and dumbbells containing chemically active acylphosphate internucleotide groups were synthesized. To obtain these compounds the chemical ligation method was used. The acylphosphate group was inserted into a DNA duplex and dumbbells as a result of template-directed condensation of 5'-phosphate and especially introduced 3'-carboxy groups of oligonucleotides. 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC) was used as a condensing agent. Oligonucleotides containing a carboxy group were obtained by the interaction of their 3'-phosphate with glycine methyl ester under the action of EDC, followed by ester hydrolysis. The yields of acylphosphate-containing double-stranded oligonucleotides achieved 15-25% depending on the structure of their precursors. It was shown that these compounds are acylating agents and are efficiently cleaved in near-physiological conditions under the action of ethylenediamine or N-methylimidazole. These results indicate that double-stranded oligonucleotides carrying acylphosphate internucleotide groups could constitute new crosslinking reagents for affinity modification of DNA recognizing proteins.