The quinobenzoxazines are a group of topoisomerase II catalytic inhibitors that have demonstrated promising anticancer activity in mice. They have been proposed to form an unprecedented 2:2 drug-Mg2+ self-assembly complex on DNA. We have exploited the photochemical decomposition of the quinobenzoxazines to gain further support and insights into the nature of 2:2 quinobenzoxazine-Mg2+ dimers and the 2:2 drug-Mg2+ complex on duplex DNA. The quinobenzoxazine A-62176 undergoes photodecomposition to highly fluorescent products. Methyl viologen (MV(2+)) accelerates this photoreaction almost 500-fold. The formation of 2:2 drug-Mg2+ dimers in solution is deduced from the Mg2+-dependent difference in the MV(2+)-facilitated photoreaction rates of racemic and scalemic A-62176. However, both racemic and scalemic A-62176 have identical MV(2+)-facilitated photoreaction rates in the presence of Mg2+ and the achiral fluoroquinolone norfloxacin, due to heterochemical norfloxacin/A-62176 dimer complex formation. DNA also accelerates the photochemical decomposition of A-62176 up to 80-fold. This DNA-acceleration requires Mg2+, duplex DNA, molecular oxygen, and intercalation of the drug into the DNA duplex. In the proposed model for drug-DNA complexation, only one drug molecule of each 2:2 drug-Mg2+ dimer intercalates into the DNA duplex, the other molecule binds externally to the DNA. Norfloxacin, which can only play the external binding role, was able to modulate the photochemical reaction of the quinobenzoxazines on DNA. Furthermore, it appears that the precise positioning of the intercalated molecule, which is modulated by the structure and stereochemistry of the externally bound molecule, plays an important role in determining the rate of photoreaction on DNA. The implications of the observed photochemical reaction of the quinobenzoxazines are described for human phototoxicity, photodynamic therapy, mechanism of action studies, and improved drug design for both topoisomerase and gyrase inhibitors.