We developed an assay that utilizes electrospray ionization mass spectrometry (ESI-MS) to determine rapidly the noncovalent binding of drugs with oligodeoxynucleotides and to assess their relative affinities and stoichiometries. The method uses a set of self-complementary oligodeoxynucleotides that differ in length (6-mer to 12-mer), motif (GC-rich or AT-rich). and sequence, and these were annealed to form duplexes. To the oligodeoxynucleotides are bound a group of drugs (distamycin, Hoechst 33258, Hoechst 33342, berenil and actinomycin D abbreviated as D1, D2, D3, D4, and D5, respectively) that are classic minor-groove binders and intercalators. A second group (porphyrin H(2)TMpyP-4, metalloporphyrin CuTMpyP-4, FeTMpyP-4 and MnTMpyP-4, and [Ru(II)12S4dppz]Cl-2 abbreviated as D6, D7, D8, D9, and D10) binds via mixed modes (i.e., groove binders and intercalators). The results confirm the binding stoichiometry and show preferred binding of minor-groove binders (distamycin, Hoechst 33258, Hoechst 33342, and berenil) to AT-rich oligomers and preferred interaction of the intercalator actinomycin D with CC-rich oligomers. The drugs H2TMpyP-4 and CuTMpyP-4 bind via mixed modes, whereas FeTMpyP-4 and MnTMpyP-4 interact by minor groove-binding only. Competitive binding experiments show that group-I drugs with duplex 5'-CGCAAATTTGCG-3' have binding affinities in the order D3 > D2 > D1 > D4. The order for group-II drugs with duplex 5'-ATATAT3-' is D6 approximate to D7 > D8 approximate to D9.