The collision-induced dissociation (CID) fragmentation reactions of a variety of deprotonated peptides containing proline have been studied in detail using MS2 and MS3 experiments, deuterium labelling and accurate mass measurements when necessary. The [MHCO2]− (a2) ion derived from H-Pro-Xxx-OH dipeptides shows an unusual fragmentation involving loss of C2H4; this fragmentation reaction is not observed for larger peptides. The primary fragmentation reactions of deprotonated tripeptides with an N-terminal proline are formation of a3 and y1 ions. When proline is in the central position of tripeptides, a3, y2 and y1 ions are the primary fragmentation products of [MH]−, while when the proline is in the C-terminal position, a3and y1 ions are the major primary products. In the latter case, the a3 ion fragments primarily to the ″b2 ion; further evidence is presented that the ″b2 ions have a deprotonated oxazolone structure. Larger deprotonated peptides having at least two amino acid residues N-terminal to proline show a distinct preference for cleavage of the amide bond N-terminal to proline to form, mainly, the appropriate y ion. This proline effect is compared and contrasted with the similar proline effect observed in the fragmentation of protonated peptides containing proline. Copyright © 2005 John Wiley & Sons, Ltd.