The charge state distribution and CID fragmentation of two series of deprotonated oligodeoxynucleotide (ODN) 9-mers (5=-GGTTXTTGG-3= and 5=-CCAAYAACC-3=, X/Y ϭ G, C, A, or T) have been studied in detail in an ion trap in an effort to understand the intrinsic properties of DNA in vacuo. The distribution of charge states (Ϫ2 to Ϫ6) is similar for both the X-and Y-series, with the most abundant being the Ϫ4 charge state. The T-rich X-series prefers higher charge states (Ϫ6 and Ϫ5) than does the Y-series. Calculations show that phosphate groups located nearest a thymine are more acidic than those near an adenine, cytosine, or guanine, thus explaining why the X-series prefers higher charge states. We use the term "charge level" to define the ratio of the charge state to the total number of phosphate groups present in the ODN. We find, consistent with previous studies, that the initial step of fragmentation is loss of nucleobase either as an anion or as a neutral. We observe the former for ODNs with charge levels greater than 50% and the latter for ODNs with charge levels below 50%. The overall anionic base loss follows the trend A Ϫ Ͼ Ͼ G Ϫ Ϸ T Ϫ Ͼ C Ϫ ; electrostatic potential calculations indicate that this trend follows delocalization of electron density for each anion, with A Ϫ being the most stabilized through delocalization. For neutral base loss, thymine (TH) is rarely cleaved, while the preferences for AH, GH, and CH loss vary. Proton affinity (PA) calculations show that a nearby negatively charged phosphate enhances the PA of proximally located nucleobases; this PA enhancement probably plays a role in promoting neutral base loss. The trends differ by charge level. At a charge level of 37.5% (Ϫ3 charge state), AH loss is preferred over CH and GH loss, regardless of sequence. However, at a charge level of 25% (Ϫ2 charge state), the terminal bases are preferentially lost over the internal bases, regardless of identity. By reconstructing the ODN sequences from structurally informative (a-BH) and w ions, we are able to identify the charge locations for the Ϫ3 and Ϫ2 charge states. For the Ϫ3 charge state, one charge resides on each "most terminal" phosphate, with the third being in the middle. For the Ϫ2 charge state, each charge resides on the penultimate phosphate groups. We compare our data to earlier experiments in an effort to generalize trends. (J Am Soc Mass Spectrom 2005, 16, 1853-1865