Previous studies have defined specific functional relationships within monophosphoryl lipid A (MLA) preparations. To extend this understanding to all contributing entities, MLA samples have been structurally characterized using electrospray ionization, collision-induced dissociation (CID), and tandem mass spectrometry (MS/MS). MLA profiles of Salmonella minnesota Re595 have been compared with Shigella flexneri for sample type and component distribution. In excess of 20 individual structures compose each sample, which differ considerably in abundance but little in composition. Component heterogeneity can be directly related to alkane chain length, "lipid X"-type analogs, and variations in esterification to the core 2-amino-2-deoxydisaccharide, GlcNH2β(1-6)GlcNH2. The previously defined heptaacyl structure in S. minnesota Re595 was identified at only 15% with the most abundant species a hexaacyl analog. Profiles of S. flexneri MLA show an absence of any heptaacyl analog, with the pentaacyl component the most abundant. To confirm structural relationships, ions from both samples were fragmented by CID and separated by MS/MS. Product ion spectra proved to be identical, showing a series of acyl losses and glycosidic cleavage fragments. Since the position of each acyl group has been previously established in S. minnesota Re595 MLA, ions in the CID spectrum of S. flexneri sample could be structurally assigned. Knowledge of these structural details, their isolation, and biological testing may provide components of unique immune adjuvancy or block-selective deleterious endotoxic processes.