The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intra- and intermolecular disulfide bond was investigated using MSn tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16–31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the SS or CS bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or z-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the SS or CS bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the SS bond, the abstraction of a hydrogen atom at Cβ by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at Cα is suggested to lead to CS bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH. Copyright © 2011 John Wiley & Sons, Ltd.