To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level， it is crucial to obtain a detailed knowledge about their dynamic and structural properties. The kinetics of backbone amide hydrogen exchange is intimately linked to the structural dynamics of the protein， and in recent years， the monitoring of the isotopic exchange of these hydrogens by mass spectrometry has become a recognized method. At present， the resolution of this method is， however， limited and single-residue resolution is typically only obtained for a few residues in a protein. It would therefore be desirable if gas-phase fragmentation could be used to localize incorporated deuterons as this would ultimately lead to single-residue resolution. A central obstacle for this approach is， however， the occurrence of intramolecular migration of amide hydrogens upon activation of the gaseous protein (i.e.， hydrogen scrambling). Here we investigate the occurrence of scrambling in selectively labeled peptides upon fragmentation by matrix-assisted laser desorption/ionization in-source decay (MALDI ISD). We have utilized peptides with a unique regioselective deuterium incorporation that allows us to accurately determine the extent of scrambling upon fragmentation. Our results show that the level of scrambling upon MALDI ISD is so low that the solution deuteration pattern is readily apparent in the gas-phase fragment ions. These results suggest that MALDI ISD may prove useful for hydrogen exchange studies of purified peptides and small proteins.