The aim of this study was to define the optimal conditions for the plasma pretreatment and to improve the production of standard curves for plasma endotoxin determination by a chromogenic substrate assay. Endotoxin standard from E. coli O 111:B 4 (0-50 ng/l) was added to pyrogen-free water or to plasma samples from 12 healthy subjects and 24 alcoholics, before pretreatment by heating (75 degrees C, 5 minutes) or with perchloric acid (0.32 mol/l). When endotoxin standard curves were determined using a microprocessor-controlled reader, the slopes of the curves obtained with plasma differed from those with pyrogen-free water. The slope of the standard curve prepared with plasma samples from different patients exhibited marked interindividual variations. Compared with the heating method, the perchloric acid method gave more variable results and a lower recovery of added endotoxin, especially in plasma from alcoholics. The results permit the following conclusion: 1. For plasma endotoxin determination, a standard curve should be prepared for each individual plasma sample. 2. The endotoxin standard should be added before pretreatment of the plasma. 3. Pretreatment of the plasma by heating at 75 degrees C for 5 minutes provides more reliable results than pretreatment with perchloric acid.