Eucalyptus globulus wood chips were subjected to autohydrolysis pretreatment at 175 degrees C at three different residence times. Part of the recovered solids were submitted to alkaline extraction with NaOH solution to remove leachable lignin. The chemical composition of the fibrous material was analyzed by HPLC, Py-GCMS and 2D-NMR HSQC, while morphological changes were evaluated by SEM and LSCM. The pretreated materials were hydrolyzed with cellulases at a substrate loading of 10% (w/v) for up to 72 h. Glucose yields (based on dry wood) obtained in the enzymatic hydrolysis ranged between 38% and 65%, depending on reaction time in the autohydrolysis pretreatment. After the alkaline extraction, no significant change was observed in the yields in the enzymatic hydrolysis at 72 h, but at the lower severities, the initial rates of saccharification increased. The main effect of the hydrothermal pretreatment was removal of hemicelluloses, resulting in enriched cellulose pulps. SEM and LSCM images of the hydrothermal pretreated samples showed a disruption of the fiber surface, mainly in those samples obtained at the higher severity. Py-GC/MS and HSQC analysis showed that no major changes in the lignin structure occurred in the samples obtained by autohydrolysis and further alkaline extraction. By autohydrolysis at the higher severity (So=4.02), the lateral chains in lignin were cleaved and the formation of lignin droplets was observed. Hemicelluloses removal and lignin redeposition as droplets in certain regions of the fiber surface was associated with the higher accessibility of cellulose and the yield increase of the enzymatic hydrolysis.