We found here that dynemicin A effectively breaks DNA strands under alkaline pH condition. Binding of dynemicin A to double-stranded DNA clearly interrupts the digestion reaction of exonuclease III. The DNA association sites of dynemicin A correspond considerably well to its cleavage sites. Dynemicin A seems to intercalate preferentially into the relaxed region of the DNA double helix. On the other hand, the alkali-product of dynemicin A was chromatographically identified with dynemicin N, suggesting a DNA cleavage mechanism similar to the reductant- and light-induced activation systems of dynemicin A. In order to detect drug binding to the relaxation structure in the DNA duplex, the present exonuclease III-digestion-stop-sequence method is useful.