We have investigated the site-specific cleavage of DNA by the antitumor antibiotics talisomycin and bleomycin by using 5'- or 3'-terminal 32P-labeled restriction fragments of pBR 322 DNA. Both drugs cleaved DNA preferentially at G-C and G-T sequences. However, the relative amounts of cleavage at particular cleavage sites differed between talisomycin and bleomycin at concentrations of the drugs which produced similar extents of total cleavage. In addition, talisomycin produced specific cleavages at G-A sequences which were relatively resistant to cleavage by bleomycin. Within a preferred sequence group (i.e., G-C sequences), some sites were cleaved to a greater extent relative to others by both talisomycin and bleomycin, suggesting that a greater degree of specificity than that provided by only two nucleotides is involved in the site-specific recognition and cleavage of DNA by these drugs.