Electron capture dissociation (ECD) of a series of custom-synthesized oligonucleotide pentamers was performed in a Fourier-transform mass spectrometer with a conventional filamenttype electron gun. Dissociation of oligonucleotide ions by electron capture generates primarily w/d-type and z/a-type ions with and without the loss of a nucleobase fragment ions. Minor yields of radical [z/a ϩ H]· fragment ions were also observed in many cases. It is interesting to note that some nucleoside-like fragment ions and protonated nucleobase ions (except thymine-related nucleobases and nucleoside-like fragments) were observed in most ECD spectra. The formation of these low-mass fragment ions was tentatively attributed to the secondary fragmentation of the radical S tructural characterization of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) is important, as DNA and RNA play important roles in many biochemical processes. Even with the completion of the human genome project, the importance of studying nucleic acid remains. For instance, a large region of DNA requires re-sequencing [1] to allow the detection and characterization of nucleotide mutation. There is a constant demand for powerful and rapid analytical methods to determine the structure of nucleic acids and their constituents. With the development of "soft" desorption/ionization techniques, matrix-assisted laser desorption/ionization (MALDI) [2,3] and electrospray ionization (ESI) [4,5] methods, mass spectrometry has become an indispensable, quick, and reliable tool for the analysis of oligonucleotides [6]. However, simple mass measurement provides little structural information for confirmation of the sequence for unknown natural and synthetic oligonucleotides. Much research effort has recently been devoted to the development of various tandem mass spectrometry (MS/MS) methods for structural elucidation of biomolecules. The critical event in tandem mass spectrometry of biomolecules is the activation of the selected precursor ions to induce unimolecular dissociation. Common ion activation methods used for analysis of biomolecules include lowand high-energy collision-induced dissociation (CID) [7, 8], surface-induced dissociation (SID) [9], infrared multiphoton dissociation (IRMPD) [10], and blackbody infrared radiative dissociation (BIRD) [11]. However, fragment ions observed in the tandem mass spectra using these ion activation methods mostly originate from cleavages of weak bonds [12].A relatively new ion activation method, electron capture dissociation (ECD) [13] has demonstrated several interesting features. For example, for protein-type biomolecules, ECD leads predominantly to the dissociation of the N-C ␣ linkage even in the presence of labile post-translational modifications [14,15]. A combination of ECD and other dissociation methods, such as IRMPD and CID, has been shown to provide complementary information for unambiguous identification of the type and the position of several important protein posttranslational modifications [16 -18] and for de novo s...