Interaction studies between daunomycin (DM) and enzymatically spin-labeled nucleic acid duplexes reveal two modes of binding by electron spin resonance (ESR) spectroscopy. At a low drug/nucleotide (D/N) ratio, the drug binds in the intercalative mode with only a slight reduction in base mobility. Saturation in the intercalative mode is achieved at a lower D/N ratio for B' DNA than for B DNA. After full intercalation, further addition of DM seems to destabilize the helix and to allow the formation of redox-active DM stacks complexed to the nucleic acid lattice. These stacks will irreversibly oxidize all the nitroxides covalently bound to the 4- or 5-position of the pyrimidine base. Interactions between DM and spin-labeled single-stranded nucleic acids lead directly to the formation of redox-active complexes, while mixing of the drug with spin-labeled nucleic acid building blocks not incorporated in a nucleic acid lattice causes no ESR signal change. Complete disappearance of the ESR signal of spin-labeled nucleic acids extrapolates to a D/N value which is a constant for a particular lattice system and is independent of spin-labeling content.