Endotoxin contamination of protein solutions was reduced by a phase separation technique using the detergent, Triton X-114. Protein solutions containing endotoxin were treated with Triton X-114 on ice. The solution was then warmed to 37 degrees C, whereupon two phases formed. The Triton X-114 phase, containing the endotoxin, was precipitated by centrifugation. The first cycle of phase separation produced a 1000-fold reduction of endotoxin from contaminated preparations of cytochrome c, catalase and albumin. Complete removal of endotoxin could be achieved by further cycles of phase separation. Each cycle of phase separation resulted in only a 2% loss of protein, and could be completed within 15 min. The small amount of detergent (0.018%) that persisted in protein solution could be removed by gel filtration or absorption. Proteins treated by this procedure retained normal functions. This phase separation technique provides a rapid and gentle method for removing endotoxin from protein solutions.