Highly purified and well-characterized samples of precursors and derivatives of bacterial lipopolysaccharides (LPS) were used to study the relationship between the chemical structure of lipid A and its toxicity. These samples included lipids X and Y (monosaccharide precursors of lipid A) from Escherchia coli MN7; incomplete lipid A (a disaccharide precursor of lipid A) from Salmonella typhimurium i50; and monophosphoryl lipid A, TLC-3 (a derivative of LPS), from S. typhimurium G30/C21. In addition, a diphosphoryl lipid A, TLC-3, was prepared from LPS of S. typhimurium G30/C21 and characterized by positive fast atom bombardment mass spectrometry. The diphosphoryl lipid A, TLC-3 fraction, was determined to be very toxic by the chick embryo lethal-dose test (CELD50 = 0.0064 microgram). Lipids X and Y, incomplete lipid A, and monophosphoryl lipid A (TLC-3), were all nontoxic (CELD50 greater than 10 micrograms). These results suggest a multiple structural requirement for toxicity of lipid A. Toxic lipid A must contain all of the following components: a glucosamine disaccharide, a sugar-1-phosphate, and normal fatty acid(s).