The N-terminal phenylthiocarbamoyl (PTC) derivatives of peptides and the phenylthiohydantoin (PTH) derivatives of amino acids are the two major types of products generated in the Edman protein sequencing method. Understanding the fragmentation pathways of these species should facilitate structural elucidation and chemical identiΓcation based on the fragment ion mass spectra, particularly when mass spectrometry is combined with the Edman sequencer for the analysis of non-standard and modiΓed amino acids. In this study, dissociation of the protonated PTH-X (where X = Thr, Ser, Trp and Tyr), PTH-Gly, PTC-X-Leu and PTC-Gly-Leu in electrospray ionization mass spectrometry was examined to investigate whether there is any isomerization of PTC to PTH derivatives in the gas phase during the fragmentation. It is shown that dissociation of the protonated PTH-X proceeds via hydrogen transfer from the side-chain of the amino acid to the PTH moiety with the elimination of the side-chain as a neutral species. The ions at m/z 193 formed from the source fragmentation of the protonated PTH-X are found to have the same structure and fragmentation pathways. The presence of this m/z 193 ion and its collisionally induced dissociation (CID) spectrum are unique for the PTH derivatives and they can be used to detect the presence of the PTH ions. It is shown that there is no isomerization of the thiazolone ions to the PTH ions during the dissociation of PTC-X-Leu (in this case, the ions from PTC-X-Leu are believed to have the protonated b 1 thiazolone structure). In addition, comparative studies of CID spectra of PTH-X and PTH-Gly or PTC-X-Leu and PTC-Gly-Leu are presented. The proposed fragmentation mechanisms for the protonated PTH and PTC derivatives and the m/z 193 ions are given.