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A rapid method for the separation and quantitation of unesterified cholesterol and cholesteryl esters by high-performance liquid chromatography is described. All of the cholesteryl esters typically present in plasma were resolved on Zorbax ODS reversed-phase columns using a linear gradient of water (3%-0%) in acetonitrile-tetrahydrofuran 65:35 (v/v) as eluting solvent. Results obtained by this method show good agreement with chemical and gas-liquid chromatographic methods for quantitation of cholesterol and determination of cholesteryl ester percentage compositions. High-performance liquid chromatography provides the only available method for the separation and quantitation of individual cholesteryl esters from lipid extracts of biological samples.