Several rhamnolipid preparations from Pseudomonas strains were studied by thin-layer chromatography/fast atom bombardment (TLC/FAB) mass spectrometry and TLC/FAB tandem mass spectrometry (MS/MS). The preparations were separated with normal-phase (Silica 60) and reversed-phase (RP-8) chromatography. Silica 60 plates appeared to be very useful in the separation of rhamnolipids according to the number of monosaccharide residues present. Spectra which show characteristic fragment ions could be obtained from components of mixtures with a total sample size of less than 200 ng. Chromatography on RP-8 plates gave a good separation of the rhamnolipids based on the length of the fatty acid alkyl chain. MS/MS of the sodium cationized molecules gave information about the sequence of the building blocks. Particularly, heterogeneity in beta-hydroxy fatty acid composition was determined for the principal as well as minor components present in natural rhamnolipid mixtures.