The cleavage of DNA by esperamicin is greatly accelerated in the presence of thiol compounds. Oxygen and active oxygen-radical scavengers have no significant influence upon DNA strand breakage by esperamicin. The preferential cutting sites of esperamicin are at thymidylate residues, and the frequency of bases attacked (T greater than C greater than A greater than G) is different from that of calicheamicin (C much greater than T greater than A = G), neocarzinostatin (T greater than A greater than C greater than G), or bleomycin (C greater than T greater than A greater than G). Esperamicin preferentially attacks at T and C bases in oligopyrimidine sequences such as 5'-CTC-3', 5'-TTC-3', and 5'-TTT-3'. In contrast to the preferred sites of cleavage by bleomycin, 5'-GT-3' and 5'-GC-3', the preferred sites of esperamicin-mediated DNA degradation are 5'-TG-3' and 5'-CG-3' sequences. The nucleotide-specific cleavage mode of esperamicin is significantly affected by pretreatment of DNA with netropsin and distamycin A, suggesting that interaction of esperamicin occurs through the minor groove of B-DNA. This is further supported by the asymmetric cleavage pattern to the 3' side on the opposite strand of the DNA. The roles of the fucose-anthranilate moiety and the trisaccharide side chain of esperamicin in DNA binding and base recognition are discussed.