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Non-covalent binding of actinomycin D (AMD) to single-stranded DNA (ssDNA) was observed by ion spray mass spectrometry. Interactions between AMD and different sequences of non-self-complementary oligodeoxynucleotides were investigated as a function of pH. Non-covalent AMD/ssDNA complexes disappeared at low pH, and no ssDNA complexes were detected with rifampicin (RP). Moreover, the different binding affinities between AMD and ssDNA with and without 5'G-C3' or 5'C-G3' base sequences were also demonstrated using ion spray mass spectrometry. Additional support for the non-covalent nature of binding in AMD/ssDNA complexes was obtained from tandem mass spectrometry.