A high performance gel filtration method for the rapid and reproducible separation of free and apolipoprotein D-associated lecithin: cholesterol acyltransferase (LCAT) originating from human plasma has been developed. Starting from step 3 of a previously invented covalent chromatography procedure, free LCAT was obtained as a well separated fraction in a yield of 55% of that injected into the column. The free LCAT had a specific activity of over 34000 units/mg and did not contain apolipoprotein D or any other contaminant in the injected sample. Further 28% of LCAT with fully retained activity was recovered in a second fraction, demonstrating a 66000 u LCAT associated with all apolipoprotein D occurring as a mean 33000 u and a minor 66000 u species and with at least two unidentified proteins with apparent molecular masses of 76000 u and 43000 u, respectively. Both free and apolipoprotein D-associated LCAT accepted the free cholesterol of heat-inactivated plasma selectively depleted of VLDL and LDL (α-LCAT activity) and of HDL (β-LCAT activity) as substrate.