Methylated and unmethylated CpG-ODNs at A-rich, C-rich, G-rich and T-rich conditions were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The methylted site could be confirmed by comparison of the MS/MS spectra of methylated and unmethylated CpG-ODNs. The fragmentation patterns of the CpG-ODNs were not influenced by the presence of the methyl group but significant effects were observed for nucleobase identities and parent ion charges. The cleavage at guanine was the most facile while that at thymine was the least facile. With the increase of the parent ions charge states, the major dissociation behaviors changed from the middle to the 3' and 5' termini of the sequence.