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A method has been devised for the measurement of endotoxin in plasma using the chromogenic substrate, S2222, a substrate which has been shown to be particularly sensitive to the Limulus Lysate. Time curves of the rate of release of the chromogen in mixtures in which procoagulase activation was concurrent, were complex with a lag phase which was shortened by increasing endotoxin concentrations. At a final concentration of 0.5ng/ml and 370 activation was complete within 60 minutes. The enzyme was inhibited by soya bean trypsin inhibitor but not by pancreatic trypsin inhibitor or hirudin. In the method finally adopted the lysate (25µl) was incubated with endotoxin (E.coli 026.86 Difco) and magnesium chloride (final concentration 33mM) in a total volume of 225µl. After 12 minutes preincubation 165µl of S2222(0.4mM) was added and the increase in abdorbance at 405nm over two minutes measured using an Abbott Biochromatic Analyser 100. Linear assay curves were obtained with final concentration of 0.2 to 2.0ngs endotoxin/ml with ΔOD 405/min of 0.35 at 2.0ng endotoxin/ml of final incubation mixture. ΔOD /min in control tubes were of the order of 0.02. For measurement from plasma samples, endotoxin was first extracted with chloroform. Linear calibration curves were achieved at a concentration of endotoxin of 1 to 5ng/ml of whole blood with a net OD/min at the highest concentration of 0.25.