The objective of this work was to study the effect of three operational parameters (pH, hydrauhc retention time (HRT) and growing temperature) on a semi-continuous bioreactor treating Tequila's vinasses by anaerobic digestion (AD). The response was measured through four response variables: total reducing sugars (TRS) consumption, VFA's, hydrogen and methane production. Trials were done according to a factorial design. The experimental results were studied through a multiple response optimization (MRO) analysis to find single and multiple optimums for the above-mentioned variables. Mathematical models that can describe the effect of the operational parameters on each response variable were found. In this study it is shown that hydrogen production is favored at thermophilic growth (55 degrees C), operating the reactor at a slight acidic pH range and at the higher HRT in the boundaries of the experimental region. (C) 2008 Elsevier Ltd. All rights reserved.