Fractional factorial screening design and response surface methodology were applied to optimize the entrapment of glucose oxidase in liposomes by the dehydration-rehydration vesicle (DRV) method. Phosphatidylcholine from different sources, cholesterol:phosphatidylcholine (Ch:PC) and enzyme:lipid (E:L) ratios, buffer pH, sonication frequency and trehalose concentration were the parameters selected for this study. The type of phosphatidylcholine was found to be the most important factor followed by the trehalose concentration, Ch:PC ratio, sonication frequency and E:L ratio. The pH did not play an important role in the response. By treating liposomes with trehalose, as cryoprotectant, the activity of entrapped enzyme decreased by 16%. Two of the factors (cholesterol:phosphatidylcholine and enzyme:lipid ratios) were further studied in a 3(2) central composite design. The optimized liposomal formulation with an entrapment efficiency of 24% was obtained for egg yolk PC with Ch:PC and E:L ratios of 0.95 and 14.69, respectively, at pH 6 and applying a sonication frequency of 150 W. (C) 2004 Society of Chemical Industry.