The objective of this work was study the effect of three pretreatments (alkalinization, thermical treatment, and sonication) on Tequila's stillages hydrolysis process in acidogenesis stage, through the following response variables: soluble chemical oxygen demand (CODs), total sugar and volatile fatty acids profile and the hydrogen production at the time. The stillages were subject to these pretreatments (according to a 2(3) factorial design); afterward they were transferred to a batch reactor at 35 degrees C and inoculated with an anaerobic digestor sludge. Multiple response optimization (MRO) analysis was done to find the global optimum for the response variables described above. This optimum is able to maximize simultaneously all these variables. It was found adequate to be useful hydrolyzing the organic matter present in Tequila's stillages. Mathematical models were fitted to observe the estimated effects of pretreatments on each response variable, then the MRO was applied. (C) 2007 Elsevier Ltd. All rights reserved.