We studied the one-step purification of endotoxin by hydroxyapatite chromatography using total lipids extracted from Escherichia coli by the Bligh and Dyer method. High endotoxic activity was detected in a fraction eluted from 0.5mg total lipids using an isopropanol gradient under salt-free buffer conditions. The fraction contained at least 7,000 endotoxin units and was completely free from other phospholipids. Notably, the running time of the chromatographic separation was approximately 4h, whereas purification by the conventional hot phenol-water procedure requires 60h. The procedure developed here is a simple, scalable, and rapid method for the purification of endotoxin.