The effects of the combination of immobilized lipases from Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) on the transesterification (ethanolysis) and hydrolysis of soybean oil, a heterogeneous substrate composed of different fatty acids, were investigated. The influences on the yields of conversion of the substrate molar ratio, enzyme content, and the ratio of TLL and RML in the mixture of the biocatalyst were analyzed using the central composite design and the response surface methodology. The optimal conditions for transesterification obtained were: substrate molar ratio of 7.5:1 ethanol:soybean oil; enzyme content of 25% (weight of oil); and 80% of TLL in the mixture of biocatalysts. For hydrolysis, the optimal conditions were: substrate molar ratio of 3:1 water:soybean oil; enzyme content of 25% (weight of oil); and 65% of TLL in the mixture of biocatalysts. Under the optimal conditions, the yields of conversion were 90% for transesterification and 95% for hydrolysis. Time courses of the reactions showed that when using the optimal mixture of lipases, the yields were higher than those obtained using only one of the enzymes, approximately 15% higher than using only TLL and more than twice than using only RML. Enzyme activities remained unaltered for both transesterification and hydrolysis, even after ten reaction cycles in which the immobilized enzymes were washed with n-hexane at the end of each batch. The use of a mixture of immobilized lipases seems to be a promising technology in order to improve the enzymatic synthesis of biodiesel and hydrolysis of oils. (C) 2010 Elsevier Ltd. All rights reserved.