Magnetic microspheres were prepared by suspension polymerization of glycidyl methacrylate, ethylene glycol dimethacrylate and vinyl acetate in the presence of oleic acid-coated magnetite, and then, alcoholysed to improve their hydrophilicity. Candida rugoso lipase was covalently immobilized on the hydrophilic magnetic microspheres via the active epoxy groups with the activity yield up to 64.2%. The resulting immobilized lipase had better resistance to pH and temperature inactivation in comparison to free lipase, the adaptive pH and temperature ranges of lipase were widened, and it exhibited good thermal stability and reusability. The resistance of immobilized lipase against urea denaturation was also improved, and the immobilized lipase retained 40% of the activity in the presence of 5 M urea whereas free enzyme retained only 8% of activity. Kinetic parameters were also determined for both immobilized and free lipase. The K-m value for immobilized (1.03 mg/ml) was higher than that of the free lipase (0.52 mg/ml), whereas the V-max value was smaller for the immobilized lipase. (C) 2008 Published by Elsevier Ltd.