Lipase (EC 22.214.171.124) from Candida rugosa was immobilized with DEAE-Sephadex A50, Sephadex G50, Sephadex LH-20, Amberlite IRA94, and Amberlite XAD-7. The enzyme immobilized with DEAE-Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (isooctane) containing olive oil substrate was cocurrently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half-life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30-degrees-C. The reactor was also operable with n-hexane, but the operational stability of the immobilized enzyme in n-hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two-phase continuous reaction in packed-bed column reactor without any surfactant supply or prior dispersion of the two immiscible phases.