An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. Cholesterol esters are hydrolyzed to free cholesterol by cholesterol ester hydrolase (EC 22.214.171.124). The free cholesterol produced is oxidized by cholesterol oxidase to cholest-4-en-3-one with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to form yield a red colored quinoneimine dye with maximum absorption at 500 nm. The method is reproducible, and the results correlate well with those obtained by automated Liebermann-Burchard procedures (AA-2 and SMA 12/60) and the method of Abell et al. The present method affords better specificity than those previously reported and has excellent precision.