Reaction of the anthracycline, antitumor drugs adriamycin and daunomycin with the self-complementary DNA oligonucleotide GCGCGCGC, (GC)4, in the presence of the reducing agent dithiothreitol, the oxidizing agent hydrogen peroxide, or the alkylating agent formaldehyde gives a similar mixture of DNA-drug adducts. Negative ion electrospray mass spectra indicate that adduct formation involves coupling of the DNA to the anthracycline via a methylene group and that the major adduct is duplex DNA containing two molecules of anthracycline, each bound to a separate strand of the DNA via a methylene group. The source of the methylene group is formaldehyde. A molecular structure with each anthracycline intercalated at a 5'-CpG-3' site and covalently bound from its 3'-amino group to a 2-amino group of a 2'-deoxyguanosine nucleotide is proposed based upon spectral data and a relevant crystal structure. The reaction of (GC)4 with the anthracyclines and formaldehyde forms an equilibrium mixture with DNA-drug adducts which is shifted toward free DNA by dilution. The results suggest a pathway to the inhibition of transcription by reductively activated adriamycin and daunomycin. Reductive activation in the presence of oxygen yields hydrogen peroxide; hydrogen peroxide oxidizes constituents in the reaction mixture to formaldehyde; and formaldehyde couples the drug to DNA. In this regard, hydrogen peroxide reacts with adriamycin via Baeyer-Villiger reactions at the 13-position to yield 2, 3, and formaldehyde. Formaldehyde also results from hydrogen peroxide oxidation of Tris [tris(hydroxymethyl)aminomethane] present in transcription buffer and spermine, a polyamine commonly associated with DNA in vivo, presumably via the Fenton reaction.