Characterization of epsilon-N-acetylated lysine containing peptides, one of the most prominent post-translational modifications of proteins, is an important goal for tandem mass spectrometry experiments. A systematic study for the fragmentation reactions of b ions derived from epsilon-N-acetyllysine containing model octapeptides (K(Ac)YAGFLVG and YAK(Ac)GFLVG) has been examined in detail. Collision-induced dissociation (CID) mass spectra of b(n) (n=4-7) fragments of epsilon-N-acetylated lysine containing peptides are compared with those of N-terminal acetylated and doubly acetylated (both epsilon-N and N-terminal) peptides, as well as acetyl-free peptides. Both direct and nondirect fragments are observed for acetyl-free and singly acetylated (epsilon-N or N-terminal) peptides. In the case of epsilon-N-acetylated lysine containing peptides, however, specific fragment ions (m/z 309, 456, 569 and 668) are observed in CID mass spectra of b(n) (n=4-7) ions. The CID mass spectra of these four ions are shown to be identical to those of selected protonated C-terminal amidated peptides. On this basis, a new type of rearrangement chemistry is proposed to account for the formation of these fragment ions, which are specific for epsilon-N-acetylated lysine containing peptides. Consistent with the observation of nondirect fragments, it is proposed that the b ions undergo head-to-tail macrocyclization followed by ring opening. The proposed reaction pathway assumes that b(n) (n=4-7) of epsilon-N-acetylated lysine containing peptides has a tendency to place the K-Ac residue at the C-terminal position after macrocyclization/reopening mechanism. Then, following the loss of CO, it is proposed that the marker ions are the result of the loss of an acetyllysine imine as a neutral fragment. Copyright (c) 2014 John Wiley & Sons, Ltd.