The effects of changes in the conformational state of DNA on the single-strand and double-strand breakage activity of two antitumor antibiotics, bleomycin (BLM) A2 and phleomycin D1, have been studied by the gel electrophoretic analysis of the drug-degraded PM2 phage superhelical DNA pretreated with an intercalating agent, ethidium bromide (EB). Both the single-strand and double-strand breakage activities of BLM A2 increased as the negatively superhelical turns of native PM2 DNA were gradually removed by intercalation with increasing EB concentrations. The activities peaked when DNA was completely relaxed and gradually decreased as the higher concentrations of EB twisted DNA into the positively superhelical form. The decrease in breakage activity was not due to any inhibitory effect of EB at higher concentrations, since treatment of the relaxed Form I0 DNA with low EB concentrations also reduced the activity. In contrast to BLM A2, phleomycin D1 responded minimally to DNA conformational changes, which suggested further that the two drugs may react with DNA differently. The differential responses of BLM A2 activity towards different DNA conformational states may have biological implications, since DNA in cells may exist in different conformational states relating to various gene functions. The current study may serve as a model for studying combined effects of intercalative and nonintercalative antitumor antibiotics which are used frequently in combination treatments of cancer.