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An accurate yet simple procedure for the determination of total cholesterol, based upon the application of a Liebermann-Burchard color reaction directly in the solvent employed for extraction of cholesterol from serum, has been described. Extraction of cholesterol and removal of protein are accomplished by means of acetic acid and acetic anhydride. Serum water is removed by the acid-catalyzed hydrolysis of acetic anhydride. The Liebermann-Burchard color is then developed with a stable, modified reagent consisting of equal volumes of H2SO4 and acetic acid. Excellent agreement with the technic of Schoenheimer and Sperry is obtained. Equal intensities of color are produced by equivalent concentrations of free and esterified cholesterol. Preliminary saponification of cholesterol esters is therefore not required. Color development may proceed in ordinary room lighting without loss of accuracy.

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