Mass spectrometry of hexose-containing disaccharides often yields product ions of m/z 221 in the negative ion mode. Using a Paul trap, isolation and collision-induced dissociation of the m/z 221 anions yielded mass spectra that easily differentiated their stereochemistry and anomeric configuration, for all 16 stereochemical variants. The ions were shown to be glycopyranosyl-glycolaldehydes through chemical synthesis of their standards. The stereochemistry dramatically affected fragmentation which was dependent on four relative stereochemical arrangements: (1) the relationship between the hydroxyl group at position 2 and the anomeric configuration, (2) a cis relationship of the anomeric position and positions 2 and 3 (1,2,3-cis), (3) a 1,2 trans-2,3 cis relationship, and (4) the relationship between the hydroxyl group at position 4 and the anomeric configuration. After labeling the reducing carbonyl oxygen of a series of disaccharides with O-18 to mass-discriminate between their monosaccharide components, it was demonstrated that m/z 221 anions are comprised of an intact nonreducing sugar glycosidically linked to a 2-carbon aglycon derived from the reducing sugar, irrespective of the linkage position between monosaccharides. This enabled the location of the intact sugar to be assigned to the nonreducing side of a glycosidic linkage. Detailed studies of experimental factors necessary for reproducibility demonstrated that the unique mass spectrum for each m/z 221 anion could be obtained from month-to-month through the use of an internal energy-input calibrant ion that ensured reproducible energy deposition into the ions. The counterparts to these ions for the 2-acetamido-2-deoxyhexoses were m/z 262 anions, and the anomeric configuration and stereochemistry of these anions could also be reproducibly discriminated for N-acetylglucosamine and N-acetylgalactosamine. The fragmentation patterns of m/z 221 anions provide a firm reproducible basis for assignment of sugar stereochemistries in the gas phase.