The structural analysis of naturally occurring bacterial phospholipids in mixtures by fast atom bombardment (FAB) mass spectrometry are reported. The bacterial strains examined included several genera of actinomycetes, two strains of Escherichia coli, and one strain each of Proteus mirabilis and Pseudomonas aeruginosa. FAB mass spectrometry proved to be a useful tool for the structural identification of phospholipids in mixtures and provided stable pseudo-molecular ions and characteristic fragment ions which permitted the identification of phosphatidylethanolamine and phosphatidyl choline. Information regarding the chain length of the fatty acids, their degree of unsaturation in the chains and the presence of hydroxyl groups was also obtained. The results obtained by FAB mass spectrometry were supported by high-resolution mass spectral data, tandem mass spectrometric studies and FAB mass spectrometry of components which had been separated and partially purified by thin-layer chromatography. Each organism displayed a highly characteristic phospholipid profile suggesting the possible use of FAB mass spectrometry as a method for rapid bacterial detection and identification.