The effect of pig manure, rice straw and clay residue concentrations, as well as their interactive effects on specific methanogenic activity (SMA) at mesophilic and thermophilic conditions were investigated in this work. A central composite design and the response surface methodology (RSM) were applied for designing the anaerobic co-digestion experiments, in order to optimize conditions to enhance methane production. The results showed a significant interaction among the substrates and an enhancement of the methane production and SMA response caused by the three components. The clay residues had a positive effect to reduce the inhibition of SMA caused by high concentration of pig manure due to the ammonia nitrogen adsorbent properties of clay demonstrated in this study by the Freundlich isotherm analysis. Thus, it was corroborated the positive effect of clay as inorganic additive for stimulating pig manure anaerobic digestion. The optimum condition for mesophilic anaerobic co-digestion of pig manure, rice straw and clay mixture was obtained for SMA values of 1.31 and 1.38 gCH(4)-COD gVSS(-1) d(-1) at mesophilic and thermophilic conditions, respectively. The optimization of the SMA using RSM made possible to identify the substrate interaction effects in a concentration range with a reduced number of experiments. Besides, the model validation proved to be useful for defining optimal combination of wastes considering their anaerobic co-digestion. SMA was also a good response variable for that purpose. (C) 2014 Elsevier B.V. All rights reserved.