KSI (3-oxo-Δ5-steroid isomerase) was purified from crudeE. colilysate by countercurrent chromatography using a polymer phase system composed of polyethylene glycol 3350 and potassium phosphate (pH 7), each at 12.5%(w/w) in distilled water. Using the cross-axis coil planet centrifuge, a preparative scale separation was performed on 25mLE. colilysate containing ca 50 mg of KSI. About 40 mg of KSI was recovered at 98% purity. A small scale separation was performed onca. 3mg of15N-labeled KSI using the toroidal coil centrifuge. The present method eliminates sample loss and denaturation caused by the solid support and yields pure proteins in both preparative and analytical separations.