Partially methylated glycosides play an important role in the stereospecificity of glycolipid antigen-antibody binding reactions. A method for the structural determination of partially methylated methyl glycosides is described. The proposed method, which is an alternative to that using alditol acetates, consists in the analysis of trimethylsilyl glycoside derivatives by gas chromatography-mass spectrometry in the electron impact (EI) mode. Seven partially methylated methyl rhamnopyranosides were synthesized, then purified by high-performance liquid chromatography. After trimethylsilylation, their EI mass spectra showed characteristic fragmentation patterns allowing the determination of the position and the number of methoxy groups in the sugar ring. This method was successfully applied to the structural elucidation of partially methylated sugars contained by a quantitatively minor phenolic glycolipid antigen isolated from Mycobacterium kansasii. Moreover, the carbons involved in the glycosidic linkages of the glycolipid tetrasaccharide moiety were also identified by this method after permethylation, methanolysis and trimethylsilylation.