The apo chicken egg white riboflavin binding protein complexes several anthracycline antitumor antibiotics and their metabolites. The Kd value for three important anthracycline glycosides (adriamycin, daunomycin, aclacinomycin A) is approximately identical at 0.5 micro M. The anthracycline occupies the flavin binding site in this complex, having its D-B rings overlaying the region normally occupied by the riboflavin A-C rings, respectively. The glycoside of the anthracycline, attached to C-7 of the A ring, is exposed to the solvent; consequently, the binding protein discriminates poorly between anthracycline A ring geometric isomers. Anthracyclinones, metabolites lacking the C-7 glycoside, are bound about 10-fold more tightly. The basis for the occupancy by anthracyclines of this flavin binding site is a steric homology (both ligands contain planar, linear conjugated rings) permitting the energetically favorable displacement of water from the hydrophobic pocket. The binding protein-anthracycline complex has been used for anaerobic aqueous redox titrations. Dithionite reduction of the anthracycline glycoside provides, in a two-electron process, the 7-deoxyanthracyclinone, via reductive elimination. The bound 7-deoxyanthracyclinone is then further two-electron reduced to the bound hydroquinone. Semiquinone radical intermediates are observed transiently; these are neither stable in solution nor complexed to the binding protein. Oxidation of the hydroquinone is accomplished by several reagents (oxygen, hydrogen peroxide, ferricyanide, cytochrome c). In the case of 7-deoxydaunomycinone hydroquinone, a mixture of two products is produced upon oxidation; these are the chromo-7-deoxydaunomycinone (identical with the first 2-e- reduction intermediate) and its leuco isomer, 8-acetyl-7,8,9,10-tetrahydro-5,8,12-trihydroxy-1-methoxy-6,11-naphthacenedione (iso-7-deoxydaunomycinone). These studies provide useful information concerning the redox properties of the anthracyclines and suggest that these antitumor antibiotics may be capable of functioning as riboflavin antagonists in vivo.