Liquid chromatography/mass spectrometry using electrospray ionisation was used to analyse rhamnolipids produced by a Pseudomonas aeruginosa strain with mannitol or naphthalene as carbon source. Identification and quantification of 28 different rhamnolipid congeners was accomplished using a reverse-phase C(18) column and a 30 min chromatographic run. Isomeric rhamnolipids that were not chromatographically resolved could be identified by interpretation of their mass spectra and their relative proportions estimated. The most abundant rhamnolipid produced on mannitol contained two rhamnoses and two 3-hydroxydecanoic acid groups. The most abundant rhamnolipid produced from naphthalene contained two rhamnoses and one 3-hydroxydecanoic acid group.