Structural analyses of oligosaccharide-malononitrile derivatives were conducted by matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) analysis in positive ion mode, and by electrospray ionization collision-induced dissociation (ESI-CID) analysis in negative ion mode. The malononitrile derivatives of oligosaccharides, which were developed for separation and detection of multi-component oligosaccharides by negative ion electrospray ionization mass spectrometry (ESI-MS), were detected by positive ion MALDI with a detection limit at a 400 fmol level even without any purification procedure for the derivatized oligosaccharide mixture. The results of structural analyses of oligosaccharide-malononitrile derivatives by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were affected drastically by the choice of matrix used, and gave much more structural information than the free oligosaccharide. The effects of the malononitrile labeling group on the structural analyses of oligosaccharide-malononitrile derivatives by positive-ion MALDI-PSD analysis in reflectron mode, and by MALDI time-of-flight mass spectrometry (MALDI-TOFMS) in linear mode, are compared with those of in-source ESI-CID analysis in negative ion mode. The malononitrile derivatization method could be a powerful tool for structural analyses of oligosaccharides. Copyright © 1999 John Wiley & Sons, Ltd.