Preparations of rabbit or human leukocytes, when incubated with bacterial endotoxins (lipopolysaccharides, LPS) are stimulated to generate a procoagulant-tissue factor activity (TFa). As LPS has been shown to consist of specific repeating oligosaccharide side chains (O-antigen) linked to a central polysaccharide core region that is, in turn, linked to the lipid region of the molecule (lipid A), we have examined the biochemical requirement of the LPS necessary for generation of TFa. Using preparations of LPS from mutant strains of bacteria, which contain varying amounts of polysaccharide in relation to lipid A, we have demonstrated that activity is associated with the lipid A region of the LPS molecule. These observations have been confirmed using isolated lipid A, which is a potent stimulator of TFa, as well as a native protoplasmic polysaccharide that is both devoid of lipid A and without detectable TFa stimulatory activity. Modification of LPS by treatment with mild alkali abrogated its capacity to stimulate TFa generation. In addition, such altered preparations of LPS partially inhibit the stimulatory effect of native LPS. Similarly, treatment of LPS (or lipid A) with the antibiotic polymyxin B substantially inhibited the stimulatory effect of LPS.