The chemotherapeutic agent bleomycin (BLM) is activated by reducing agents to break isolated DNA. Paradoxically, these same reducing agents protect cellular DNA from BLM damage. To resolve this paradox, we have examined the reaction of FeIIIBLM with DNA in the presence of ascorbate. As expected, ascorbate augments FeIIIBLM-induced DNA damage. However, when ascorbate is added to FeIIIBLM prior to exposure to DNA, a redox-inactive BLM is produced in a reaction that generates the ascorbyl radical. This reaction occurs in both ascorbate-supplemented buffer and unsupplemented plasma. In buffered solution, this reaction was found to be stoichiometric; for each mole of BLM present, 6.9 mol of ascorbate was oxidized and 4.7 mol of oxygen was consumed. Iron was found to serve only as a catalyst for the reaction. These data suggest that both activation of BLM and the generation of redox-inactive BLM occur via the same reaction and that BLM-induced DNA damage depends upon BLM reaching DNA prior to its interaction with reducing agents.