Plasma desorption mass spectrometry has recently been used with success to characterize underivatized lipid A preparations: the major molecular species present give signals indicating their masses, from which probable compositions could be inferred by using the overall composition determined by chemical analyses. In the present study, plasma desorption mass spectrometry was used to compare structures in lipid A preparations isolated from several smooth and rough strains of Escherichia and Salmonella species. Preparations isolated from strains of both genera revealed considerable variation in degree of heterogeneity (number of fatty acids and presence or absence of hexadecanoic acid, phosphorylethanolamine, and aminoarabinose). Molecular species usually associated with Salmonella lipid A were found in preparations from Escherichia sp. In addition, preparations from three different batches of lipid A from one strain of Salmonella minnesota showed significant differences in composition. These results demonstrate that preparations used for biological and structural analyses should be defined in terms of their particular molecular constituents and that no generalizations based on analysis of a single preparation should be made.