Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membrane function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measure cholesterol, originally described for whole cells (Heider and Boyett  J. Lipid Res., 19:514-518; Gamble et al.  J. Lipid Res., 19:1068-1070) and serum (Huang et al.  Clin Chem., 21:1605-1608). This method has a detection limit of 0.3 microgram cholesterol. As revealed by comparison with high-performance liquid chromatography, the fluorometric/enzymatic method with biological membranes is accurate (within 4% and 8% for intestinal and hepatic plasma membranes, respectively). The assay may be completed within 3 to 4 hours and requires neither lipid extraction nor chromatographic techniques. Kinetics of the cholesterol oxidase reaction are membrane-specific, and first-order rate constants (k) are positively correlated with membrane order.