In this work, the study of viability of the grapewine stem use, alone or combined with other lignocelulosic materials, in the elaboration of specific substrates for the cultivation of Pleurotus ostreatus (Jacq. ex Fr.) Kummer, second mushroom in importance cultivated in Spain, is described. Furthermore, two different methods of preparation of the substrate and two container sizes have been considered. As for the production parameters, the best results have been provided by the substrates that combined grapewine stem with straw and with kenaf, being unfavorable the substrates based in just grapewine stem or combined with olive mill dried waste. As for the treatment applied to the materials, the immersion in water with fungicide and later pasteurization has been favoured in front of the immersion in water alone and subsequent pasteurization and thermophilic conditioning. On the other hand, the format of large bag (15 kg of substrate) provided better results that the small one (5 kg of substarte).