The extent of internal hydrogen exchange (scrambling) within multiply charged solvent-free protein ions was investigated using a small model protein. The site-specific backbone amide protection data were obtained using protein ion fragmentation in the gas phase and compared with the available NMR data. Only minimal scrambling was detected when relatively high-energy collisional activation was used to fragment intact protein ions, while low-enargy fragmentation resulted in more significant but not random internal exchange. Increased conformational flexibility of protein ions in the gas phase did not have any effect on the extent of hydrogen scrambling under the conditions of higher-energy collisional activation but resulted in totally random redistribution of labile hydrogen atoms when the protein ion fragmentation was induced by multiple low-energy collisions.