The fragmentation mechanisms of protonated triglycine and its first-gene ration dissociation products have been investigated using a combination of density functional theory calculations and threshold collision-induced dissociation experiments. The activation barrier measured for the fragmentation of protonated triglycine to the b(2) ion and glycine is in good agreement with a calculated barrier at the B3LYP/6-31++G(d,p) level of theory reported earlier [Rodriquez, C. F. et al. J. Am. Chem. Soc. 2001, 123, 3006-3012]. The b(2) ion fragments to the a(2) ion via a transition state structure that is best described as acylium-like. Contrary to what is commonly assumed, the lowest energy structure of the a(2) ion is not an iminium ion, but a cyclic, protonated 4-imidazoliclone. Furthermore, fragmentation of the b(2) to the al ion proceeds not via a mechanism that results in HNCO and H2C=C=O as byproducts, as have been postulated, but via a transition state that contains an incipient al ion and an incipient carbene. The fragmentation of a(2) to a(1) proceeds via a transition state structure that contains the al ion, CO and an imine as incipient components.