The collision-induced dissociation of protonated uracil has been studied by tandem mass spectrometry using models extensively labeled with stable isotopes, and derivatives of the kinds found in nucleic acids. Following collisional activation at 30 eV translational energy, protonated uracil dissociates through two principal pathways which do not occur in electron ionization mass spectra: (1) elimination of NH3 almost entirely from N-3, followed by loss of CO from C-4, 0(4); (2) loss of H2O, equally from 0(2) and 0(4). Elimination of HNCO, also the principal dissociation process from odd-electron molecular ions, proceeds primarily by loss of N-3, C-Z, O(2) and 10% from N-l, C-Z, 0(2). Several secondary dissociation products are formed with quantitative site specificity of skeletal atoms: C,HO+ (4-C0, C-5, C-6); H2CN+ (N-l, C-6); C2H2+ (N-l, C-5, C-6). First-step dissociation reactions are interpreted in terms of pyrimidine ring opening at likely sites of protonation after collisional activation of MH+. Collision-induced dissociation mass spectra of uracils with structural themes common to nucleic acids (methylation, replacement of 0 by S, C-5 substitution) follow analogous reaction paths which permit assignment of sites of substitution, and exhibit ion abundance changes attributed to differences in substituent basicity and electron density.