Abstract Cholesterol oxidase from Nocardia erythropolis, Pseudomonas, and Streptomyces species was active in microemulsion in which cholesterol is well solubilized. The activity was stable in nonionic microemulsions whereas in cationic and anionic microemulsions the activity decreased with time. The coupled activity test using horseradish peroxidase which is very stable in microemulsion, was modified. The activity at very low water concentration in nonionic microemulsions increased with the water content. The kinetic constants were determined: the Michaelis constant is in the range 10 to 28 m m in the microemulsions, compared to 10 to 28 μ m in buffer. The maximum velocity was reduced by a factor of 3 to 5 compared to that in buffer. Neither substrate excess nor product inhibition was detected. The preparative oxidation of cholesterol revealed the inactivation of the cholesterol oxidase by hydrogen peroxide. In contrast to glucose oxidase, hydrogen peroxide inactivated cholesterol oxidase in the absence of substrate. Catalase provides protection during the cholesterol oxidation. Microemulsions are very good media in which to perform enzyme catalyzed reactions with substrates of low water solubility. Their use for the reproducible determination of cholesterol should be examined.