Ethanol from lignocellulosic biomass (LCB), such as agricultural and forest residues, is known as the second generation (2G) ethanol. Carbohydrate fractions in LCB (cellulose and hemicellulose) are not readily available for microbial fermentation and they need to be released by additional pre-treatment and hydrolysis operations. This process is intrinsically more difficult and costly. In this study the efficiency of enzymatic hydrolysis of the species Eucalyptus urophylla and the hybrid E. urophylla x E. grandis, submitted or not to alkaline pre-treatment with green liquor, has been evaluated. The hydrolysis was carried out with a enzymatic cocktail containing aggressive cellulases, high level of ?-glucosidases and hemicellulase at 6% (g enzyme/g solid), pH 5.0, 50 g.L-1 solids for 72 h, 45 °C and 250 rpm. The pre-treatment promoted disruption of the cellular structure of the samples, allowing easier accessibility for the enzymes and demonstrating higher effectiveness of enzymatic hydrolysis. The maximum efficiency of hydrolysis (81.3%) was observed for pretreated E. urophylla.SOLABIAA