Under nonenzymatic conditions in vitro, Adriamycin appears to form interstrand cross-links with DNA over 1-2 days. This is the first report of such Adriamycin-induced interstrand cross-links in vitro. Cross-links were measured by a fluorescence based renaturation assay and also by gel electrophoresis. Both procedures revealed an increase of cross-linking with reaction time and with increasing Adriamycin concentration and a 5-6-fold enhancement in the presence of Fe3+ ions. The cross-link contains the Adriamycin chromophore, with a lambda max of 508 nm, intercalated at the GpC site of cross-linking. Maximal stoichiometry of the cross-link was one per 11-20 bp. The cross-link appears to involve adducts of the Adriamycin chromophore linked to the N2 of guanine, with no indication that N7 of guanine is involved. Given that the mode of action of Adriamycin still remains obscure, even after 20 years of clinical use, the possibility that interstrand DNA cross-links may be associated with the clinical mechanism of action of this drug should now be fully addressed.