The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase [poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase, EC 6.5.1.1]. In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-14C- and 6-3H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients. DNA ligation was slower after chemical degradation of DNA by bleomycin than after gamma irradiation. DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells. These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin. The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions. Bleomycin B2 produced considerably more DNA breaks than bleomycin A2 over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells. The chemical analogues are structurally identical except for their cationic C-terminal amine. The actual number of DNA breaks produced by bleomycin A2 or bleomycin B2, and not the concentration of bleomycin A2 or bleomycin B2 per se, determined the amount of cell killing. DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B2 than bleomycin A2.