The gas-phase structures of the protonated pentapeptide Leu-enkephalin and its main collision-induced dissociation (CID) product ions, b(4) and a(4), are investigated by means of infrared multiple-photon dissociation (IR-MPD) spectroscopy and detailed molecular mechanics and density functional theory (DFT) calculations. Our combined experimental and theoretical approach allows accurate structural probing of the site of protonation and the rearrangement reactions that have taken place in CID. It is shown that the singly protonated Leu-enkephalin precursor is protonated on the N-terminus. The b(4) fragment ion forms two types of structures: linear isomers with a C-terminal oxazolone ring, as well as cyclic peptide structures. For the former structure, two sites of proton attachment are observed, on the N-terminus and on the oxazolone ring nitrogen, as shown in a previous communication (Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2005, 127, 17154-17155). Upon leaving the ions for longer radiative cooling delays in the ion cyclotron resonance (ICR) cell prior to IR spectroscopic investigation, one observes a gradual decrease in the relative population of oxazolone-protonated b(4) and a corresponding increase in N-terminal-protonated b(4). This experimentally demonstrates that the mobile proton is transferred between two sites in a gas-phase peptide ion and allows one to rationalize how the proton moves around the molecule in the dissociation process. The a(4) fragment, which is predominantly formed via b(4), is also confirmed to adopt two types of structures: linear imine-type structures, and cyclic structures; the former isomers are exclusively protonated on the N-terminus in sharp contrast to b(4), where a mixture of protonation sites was found. The presence of cyclic b(4) and a(4) fragment ions is the first direct experimental proof that fully cyclic structures are formed in CID. These results suggest that their presence is significant, thus lending strong support to the recently discovered peptide fragmentation pathways (Harrison, A. G.; Young, A. B.; Bleiholder, B.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006, 128, 10364-10365) that result in scrambling of the amino acid sequence upon CID.