The fragmentation of small oligodeoxynucleotides using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with 355-nm radiation from the matrix 2,5-dihydroxybenzoic acid is studied. Negative ion mass spectra of the homopolymer oligodeoxynucleotides d(A)(n), d(C)(n), and d(G)(n) (n = 4, 6, 8, and 10) show substantial cleavage at N-glycosidic and phosphodiester bonds, in contrast to d(T)(n) which shows little fragmentation. The number and intensity of the fragment peaks increases significantly with the length of the parent molecules. A series of asymmetric oligodeoxynucleotides were synthesized to study this fragmentation in greater detail. In dT(4)GT(10), dT(4)G(4)T(7), and dT(7)G(4)T(4), the primary fragmentation pathway is loss of a base followed by backbone cleavage at the 3’ C-O bond of the corresponding deoxyribose. Similar fragmentation patterns were observed with dT(4)N(4)T(7) samples where N = C or A. A statistical cleavage model described the observed patterns of fragmentation well; the model yields fractions of backbone cleavage at A, C, and G of 0.13, 0.26, and 0.27, respectively. In contrast to the fragmentation observed using 2,5-dihydroxybenzoic acid as a matrix, little or no fragmentation is observed with the matrix 3-hydroxypicolinic acid. Fragmentation of oligodeoxynucleotides in UV/MALDI is thus both nucleobase and matrix dependent.