Pseudomonas aeruginosa EBN-8 mutant rhamnolipids produced on waste oils were investigated using normal-phase thin layer chromatography and fast atom bombardment mass spectrometry. Negative ion mode mass spectra yielded [M - H](-) ions and their fragment ions, which gave some indications on the sequence of rhamnolipid biosynthesis. Five rhamnolipid homologs [viz. RC(10)C(10) (m/z 503), RC(12)C(10) or RC(10)C(12) (531), RRC(10)C(8) or RRC(8)C(10) (621), RRC(10)C(10) (649) and RRC(12)C(10) or RRC(10)C(12) (677)] were detected in four rhamnolipid combinations under the different carbon sources. The prevalence of rhamnolipids was confirmed by Fourier transform infrared and one-dimensional proton nuclear magnetic resonance. We also observed some correlations between the tensioactive characteristics and structural chemistry of the rhamnolipid surfactants.