Strand scission of DNA caused by bleomycin A2 (BLM-A2) and binding of the antibiotic to DNA was studied in vitro, examining the results of sucrose density gradient centrifugation and Sephadex G100 column chromatography. BLM-A2 caused strand scission of both single and double stranded DNA of E. coli or HeLa cells at the concentration of 1.6 μg/ml, but not that of ribosomal and transfer RNAs, in the presence of 1 mM 2-mercaptoethanol. When 2-mercaptoethanol was omitted, no strand scission was observed. The action of the antibiotic was inhibited by addition of 0.1mM Cu++, Co++, Zn++, and 1mM ETDA. By dialysis of the reaction mixture, after the incubation, double stranded DNA treated with BLM-A2 was partly converted to single strand and strand scission was more markedly demonstrated. Binding of 3H-BLM-A2 to DNA was observed regardless of the presence or absence of 2-mercaptoethanol and amounts of bound BLM-A2 to single stranded DNA was about twice higher than to the double stranded. Addition of Cu++, Zn++, EDTA, or phleomycin inhibited binding of BLM-A2 to DNA but that of actinomycin, mitomycin or pluramycin did not show any effect.