We describe a fluorometric enzymatic method for determining total serum cholesterol, based on hydrolysis of cholesterol esters to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol formed, as well as that initially present, is then oxidized by cholesterol oxidase (EC 1.1.3.6) to cholest-4-en-3-one with simultaneous production of hydrogen peroxide. The latter catalytically oxidizes homovanillic acid in the presence of peroxidase (EC 1.11.1.7) to form the highly fluorescent 2,2'-dihydroxy-3,3'-dimethoxy-biphenyl-5,5'-diacetic acid. A calibration curve is constructed from data on a series of standard cholesterol solutions vs. the corresponding fluorescence change (Δf/5 min). This curve is linear up to 4.0 g of total serum cholesterol per liter of serum. The method is specific, precise, accurate, rapid, and simple, and results correlate well with those obtained by both the Liebermann-Burchard procedure and the colorimetric enzymatic method (correlation coefficients, 0.984 and 0.981, respectively)