Bleomycin (BLM), a glycopeptide antibiotic, is activated in an Fe-dependent reaction and, when O 2 is present, catalyzes the cleavage of DNA. Drug activation takes place via two pathways. In the first, Fe(II)-BLM, an S = 2 species, and O 2 react to form a diamagnetic, ternary complex, ‘oxygenated bleomycin’. The quadrupole splitting and isomer shift (ΔEq = 2.96 mm/sec, δ = 0.16 mm/sec) are suggestive of an electronic structure best described as low spin ferric bound to superoxide anion. Single electron reduction of oxygenated bleomycin yields ‘activated bleomycin’, a form of the drug that is kinetically competent to cleave DNA. As a second pathway for activation, Fe(III)-BLM reacts with peroxide; Fe(II)-BLM is not produced as an intermediate. Activated bleomycin is an S = 1 t f species with a well resolved EPR spectrum ( g= 2.26, 2.17, 1.94). When prepared with 57Fe, the g = 1.94 EPR feature is split by 22 gauss, demonstrating that the electron spin resides primarily on the iron. When activated bleomycin is prepared from Fe(II)-BLM and 17O 2, the EPR spectrum is broadened, demonstrating the presence of at least a single oxygen atom derived from O 2. Concomitant with DNA cleavage, activated bleomycin decomposes to form Fe(III)-BLM, an S = 1 t species with EPR features (g = 2.45, 2.18, 1.89) and Mössbauer parameters (ΔEq = 2.85 mm/sec andδ = 0.20 mm/sec) closely resembling those of cytochrome P−450. Fe(III)-BLM, in the presence of phosphate or arsenate, or at pH below 4.5, becomes high spin with g eff = 4.3 at 77 °K (E/D ∼ 0.3). Yet, Fe(III) remains bound to the drug. The bonding of iron to BLM is affected by the presence of DNA. Changes are observed in the g =1.94 EPR feature for the activated complex, and in the hyperfine interaction with 14N in the low spin ferric complex, as determined from the differences in the electron spin echo spectrum. DNA also prevents the conversion of the iron in the low spin ferric complex to an S = 5 t species by phosphate or arsenate.