The sequence specificity of daunomycin was assessed using competition equilibrium dialysis, DNAse I footprinting and an E. coli RNA polymerase transcription inhibition assay; similar studies were performed on adriamycin and a new bis-intercalating daunomycin dimer. The results clearly demonstrate that the highest affinity sites are CA for daunomycin and adriamycin, and CACA for the bis-daunomycin. Other modest affinity (GC, CG, CT, TC, CC, AC) and poor affinity binding sites (AA, AT, TA) were also observed. Our results are in agreement with (a) the observed 5'-pyrimidine-purine-3' sequence preference of intercalating drugs, (b) the reported role played by OH(9) of daunomycin in the stabilization of the drug/DNA intercalation complex, and (c) the thermodynamics of nearest neighbour base-pair unstacking at the intercalation site. The CA specificity of daunomycin and adriamycin suggests that their biological activity may arise from association with the CA containing sequences which are thought to be associated with genetic regulatory elements in eukaryotes. The implications for future anthracycline drug design are presented in this context.