A negative ion electrospray ionization tandem mass spectrometric technique was developed for the analysis of glycerophospholipids. Examination of the product ion mass spectrum of the deprotonated molecular ion provided sufficient information to identify both the class of glycerophospholipid and the molecular weights of the two fatty acid moieties. This technique was applied to the profiling of glycerophospholipids present in the chloroform/methanol extracts of four different bacterial species. The principal bacterial phospholipids detected by this technique were phosphatidylglycerols and diphosphatidylglycerols, accompanied by small amounts of phosphatidylethanolamines for two of the bacterial species examined. The fatty acid composition of the phosphatidylglycerols for each bacteria was determined by tandem mass spectrometry and presented graphically. Differences in the fatty acid composition for each bacterial species were readily apparent from a visual examination of the data sets.