Sixteen monophosphoryl Lipid A (MLA) homologs obtained from the lipopolysaccharides of Salmonella minnesota Re595 were separated by preparative thin layer chromatography into eight fractions. The components of these fractions were analyzed directly (or as structural analogs) and characterized by mass spectrometry. Molecular weights were determined by negative and positive ion fast atom bombardment mass spectrometry and component structures were assigned following a study of fragmentation and metastable ion kinetic energy spectrometry. One fraction (TLC-8) contained a single heptaacyl MLA of Mr = 1,954, a structure previously elucidated (Qureshi, N., Mascagni, P., Ribi, E., and Takayama, K. (1985) J. Biol. Chem. 260, 5271-5278). The remaining seven fractions contained 15 additional MLAs with decreasing acylation. Two of these components have been previously reported in S. minnesota and Salmonella typhimurium. Three of the eight TLC fractions (TLC-8, -7, -6) were found to be biologically active toward human platelets inducing their aggregation and secretion of serotonin. All tested fractions induced varying degrees of phosphorylation of a platelet protein of Mr = 47,000 (P47) reflecting protein kinase C activation (Grabarek, J., Her, G. R., Reinhold, V. N., and Hawiger, J. J. (1990) J. Biol. Chem. 265, 8117-8121).