Cholesterol and cholesteryl esters in human macrophages were analyzed by three different methods. Values obtained by high-performance liquid chromatography and by gas-liquid chromatography were compared with those obtained using enzymatic fluorometry. We also assessed fractional lipid recovery from these cells using radiolabeled cholesterol and cholesteryl ester. Enzymatic fluorometry substantially underestimated cellular cholesterol content. Two reasons for this were found. First, recovery into a variety of solvents was incomplete, particularly when extracted lipids were dried and redissolved in a second solvent. Second, the cells appeared to contain an intrinsic inhibitor of the enzymatic fluorometric method.