A conventional limulus test is not specific to endotoxin because of the presence in amebocyte lysate of a (1----3)-beta-D-glucan-sensitive factor. By fractionating coagulation enzymes in the lysate and recombining only those factors involved in endotoxin-induced coagulation, we have developed a new test specific to endotoxin. The recombined enzymes reacted only with endotoxin, and not with fungal polysaccharides. Conventional amebocyte lysate, on the other hand, reacted with both of them. A good linearity was obtained with this method between endotoxin concentration and absorbance with a sensitivity of 1 pg/ml of Escherichia coli 0111:B4 endotoxin. The regression lines for different types of endotoxins were parallel to one another. For the correct diagnosis of endotoxemia, this new test has a definite advantage over the one using whole amebocyte lysate.