The fragmentation of protonated tripeptides under metastable ion conditions and collision-induced conditions are reported. The majority of protonated tripeptides cleave at the C-terminal amide bond to form b(2) and y(1) ions, with the relative amounts depending on the proton affinities of the amino acids derived from the C-terminal amino acid residue. Protonated tripeptides that have a proline residue at the C-terminal position fragment almost exclusively by cleavage of the amide bond adjacent to the proline and give mainly y(1) ions; where there is a proline residue in the central position, fragmentation of the protonated tripeptide occurs at both the N-terminal and the C-terminal amide bonds to form Y-2 and b(2) ions-, finally, two of the tripeptides with proline at the N-terminal position, Pro-Pro-Pro and Pro-Gly-Gly, fragment largely by cleavage at the N-terminal, the former by cleavage of the amide bond and the latter by direct formation of the a, ion. Protonated Pro-Val-Gly fragments to give predominantly b(2) and a, ions at low energy, but the a, ion becomes the dominant product at higher energies. In the fragmentation of protonated peptides, the lack of b ions formed by cleavage at the amide bond C-terminal to proline (i.e., with the proline residue in the oxazolone ring) has conventionally been attributed to ring strain in a bicyclic oxazolone. Here we show, however, that the bicyclic oxazolone structure containing proline derived front protonated Gly-Pro-Gly is only 2.7 kcal/mol higher in free energy, at the B3LYP/6-31++G(d,p) level of theory, than the isomeric nonbicyclic oxazolone derived from protonated Pro-Gly-Gly; that is, strain appears to be a small or negligible factor. Formation of the Y-2 ion in protonated Gly-Pro-Gly has a barrier of 3 kcal/mol higher free energy than that of the b ion. In comparison, the difference increases to 10 kcal/mol in protonated Gly-Phe-Gly. The neutral products that are formed along with the Y-2 ion are CO and methanimine, not aziridinone as once proposed.