Pretreatment of animals with butyrylcholinesterase (EC 3.1.1.8 BChE) provides complete protection from the acute effects of organophosphorus nerve agents. Butyrylcholinesterase has also been shown to protect from cocaine toxicity. Large amounts of highly purified butyrylcholinesterase are needed to test the effectiveness of this new therapeutic agent in monkeys. Only a minimum amount of endotoxin can be present in a therapeutic intended for injection into monkeys. Our goal was to develop a large scale purification method for human BChE from human plasma with precautions to minimize endotoxin content. A protocol was developed that processed up to 100 L of human plasma at a time. Dialysis in pH 4.0 buffer, ion exchange chromatography at pH 4, affinity chromatography on procainamide-Sepharose, and HPLC ion exchange at pH 7.4 yielded highly purified human BChE containing a low endotoxin level of about 800 EU/ml. The purified BChE produced by this method had a mean residence time of 56 h in mice and 93 h in monkeys, and caused no toxic effects. The absence of a toxic effect in monkeys demonstrates that the endotoxin level of 800 EU/ml was well tolerated by monkeys.