Computer analyses of DNA sequencing data obtained using various restriction fragments of pBR 322 DNA indicate that a trinucleotide sequence (-Pyr-G-C-) is the most preferred site for cleavage by the antitumor antibiotic bleomycin A2. Talisomycin A, a structurally related bleomycin analog, cleaved at the sequences -G-T/A- most preferentially. However, the presence of a pyrimidine at the 5' side of guanine at the cleavage site did not increase the probability of that site being cleaved by talisomycin. Using denaturing and nondenaturing polyacrylamide gel analyses of the drug-DNA reaction products. The sites of both single- and double-strand breaks have been localized and differentiated. The results indicate that a major determinant for location of a site-specific double-strand break is the production of two closely spaced sequence-specific single-strand breaks by the drugs on opposite strands of the DNA. A four-base pair sequence is proposed for the optimal sequence for bleomycin-induced double-strand breaks.