Noncovalent complexes of DNA and Hoechst 33258 were investigated by ESI-FT/ICR MS in various activation modes (collision-induced dissociation (CID), sustained off-resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton dissociation (IRMPD) and electron detachment dissociation (EDD)). The binding selectivity of Hoechst 33258 was confirmed by the comparative study of its noncovalent association with different DNA sequences. The CID spectra of [ds + HO - 5H](5-) obtained with a linear hexapole ion trap resulted in unzipping of the strands. This outcome is a clue to the drug-binding mode, shading light on the localization of the binding sites of Hoechst 33258 to the DNA sequence. The IRMPD and SORI-CID experiments mainly gave DNA backbone cleavages and internal fragment ions. From this result, information on the localization of the binding sites of Hoechst 33258 in the DNA sequence was obtained. No sodium cationization was observed on the DNA sequence ions although they were present on fragmentation of the duplex, indicating that the backbone cleavages were generated from the single strand associated with the Hoechst 33258 where the number of alkali cation is restricted. Under electron detachment (ED) conditions, multiple EDs were achieved for the [ds + HO - 5H](5-) ion without any significant dissociation. The presence of drug appears to enhance the stability of the multiply charged system. It was proposed that the studied noncovalent complex involved the formation of zwitterions and consequently strong salt-bridge interactions between DNA and drug.