In the assay for total cholesterol in lipid extracts by enzymatic methods, it was found necessary to redissolve the lipid prior to the reagent addition. Isopropyl alcohol was found to be best at promoting the colorimetric reaction. As little as 1.45 micrograms free cholesterol and 1.8 micrograms esterified cholesterol could be determined after 15 min incubation with the isopropyl alcohol-reagent. Using a formula for the correction of pigment absorbance, it was also possible to apply the enzymatic kit method to red blood cell lipid extracts.