Cholesterol esterase (sterol-ester acylhydrolase, EC 3.1.1.13) has been purified from porcine pancreas by two methods, one of which was previously reported by Momsen, W.E. and Brockman, H.L. (Biochim. Biophys. Acta. 486 (1977) 102-113). Multiple forms of the enzyme were demonstrated throughout the course of both purification procedures. These forms hydrolyzed both p-nitrophenyl acetate as well as cholesteryl oleate. Isoelectric focusing was used to select one form of cholesterol esterase having a pI of 4.3 for further study. Using high-pressure liquid chromatography on a TSK Spherogel column this apparently homogeneous preparation of cholesterol esterase was separated into two components having molecular weights equal to 90 000 (peak I) and 45 000 (peak II). The number of each amino acid residue in peak I was double that of the corresponding residue in peak II, suggesting a dimer-monomer relationship. The N-terminal analyses showed that the first five amino acid residues were the same in peak I and peak II. The enzyme is a glycoprotein containing glucosamine, glucose, galactose, mannose and rhamnose; it is inhibited by diisopropyl fluorophosphate.