The potent anticancer drug daunorubicin binds to DNA by the process of intercalation. Formaldehyde (HCOH) was found to rapidly and efficiently cross-link the drug to DNA in solution in a reaction the rate of which was strongly dependent upon HCOH concentration. The cross-linked drug remains intercalated into DNA, as judged from the results of absorbance, fluorescence, and circular dichroic spectroscopic studies and thermal denaturation studies. Comparative studies using a series of anthracycline derivatives showed that the 3'-NH2 group on the daunosamine moiety is absolutely required for cross-linking. Comparative studies using synthetic deoxypolynucleotides of defined sequence showed that the N2 amino group of guanine is absolutely required for cross-linking. In restriction enzyme inhibition assays using pBR322 DNA as a substrate, cross-linked daunorubicin was found to completely inhibit cutting by Nae I (recognition site 5′GCCGGC3′) but not by Dra I (recognition site 5′TTTAAA3′). These results (a) extend, into solution, previous reports of the cross-linking of daunorubicin to oligonucleotides in crystals; (b) show that daunorubicin can be chemically cross-linked to natural DNA samples as well as to poly- and oligonucleotides, and (c) demonstrate the base- and regioselectivity of the cross-linking reaction.