The unexplained occurrence of thrombocytopenia in cases of Gramnegative sepsis in man led us, in the light of animal experiments indicating the blood platelet as the target cell for endotoxin, to examine the effect of Salmonella enteritidis lipopolysaccharide B on human platelets. Human platelets were separated from a coat of plasma proteins by Sepharose 2B filtration or by a combined procedure of albumin gradient and Sepharose 2B filtration. The action of endotoxin on human platelets resulted in membrane changes manifested by dose-dependent release of [3H]serotonin and adenine nucleotides. Cytoplasmic marker, lactic dehydrogenase, and lysosomal marker, beta glucuronidase, were retained indicating that the release reaction was selective. Release of [3H]serotonin was specific for endotoxin since other particulates, zymosan and erythrocyte stroma, were without effect. Endotoxin, added to gel-filtered human platelets, induced a significant evolution of platelet factor 3 procoagulant activity. Preincubation of endotoxin with a membrane-rich homogenate of human platelets inhibited its labilizing effect on human platelets thus suggesting an interaction between endotoxin and the platelet membrane itself. Other plausible factors in this interaction [fibrinogen, adenine nucleotides, thrombin, sialic acid residues, and IgG] were eliminated on the basis of a series of control experiments. From the negligible effect of aspirin and indomethacin, we may infer that the interaction of endotoxin with platelets does not depend on the platelet prostaglandin synthesis pathway. The direct interaction of endotoxin with the human platelet membrane comprises a new mechanism which may help to clarify the pathogenesis of vascular and haemostatic disorders accompanying bloodstream infections due to Gram-negative bacteria.