Neutral cholesterol esterase from cytosol of porcine liver tissue was isolated by means of ion-exchange chromatography on DEAE- and CM cellulose and gel filtration on Sepharose 6B. Specific activity of the isolated cholesterol esterase using cholesteryl-14C-oleate as substrate was 61.2 nmole/mg/hr. Total activity of the enzyme was increased during ion-exchange chromatography as a result of removing of possible inhibitors or endogenic lipids. The enzyme was separated by means of gel filtration on Sepharose 6B into 3 active forms with molecular masses, approximately 200,000, 60,000 and 30,000 daltons. Cholesterol esterase from porcine liver tissue was distinct from the pancreatic enzyme by its ability to hydrolyze substrate in absence of bile acids.