Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of cleavage of complexes under the action of ultrasound. The specificity of ultrasound-induced cleavage of the DNA sugar-phosphate backbone was studied in actinomycin D (AMD) complexes with double-stranded DNA restriction fragments. After antibiotic binding, the cleavage intensity of phosphodiester bonds between bases was shown to decrease at the chromophore intercalation site and to increase in adjacent positions. The character of cleavage depended on the sequences flanking the binding site and the presence of other AMD molecules bound in the close vicinity. A comparison of ultrasonic and DNase I cleavage patterns of AMD-DNA complexes provided more detail on the local conformation and dynamics of the DNA double helix in both binding site and adjacent regions. The results pave the way for developing a novel approach to studies of the nucleotide sequence dependence of DNA conformational dynamics and new techniques to identify functional genome regions.