Biomass conversion processes have become increasingly important to mitigate fossil fuel consumption and to increase the contribution of renewable fuels into the world energy matrix. In this study, Eucalyptus urograndis wood chips were pretreated by autocatalytic steam explosion to produce cellulosic ethanol after enzymatic hydrolysis and fermentation. These experiments were organized in a central composite rotatable design using temperatures and reaction times ranging from 174 to 216 degrees C and from 4 to 11 min, respectively. Mass yields, cellulose degree of polymerization, and glucose yields after enzymatic hydrolysis showed a linear correlation with pretreatment severity. The best condition was set at 210 degrees C for 5 min due to its higher glucose yield after pretreatment and enzymatic hydrolysis. Pectins were almost completely solubilized after pretreatment while galactoglucomannans were more resistant to acid hydrolysis than arabinoglucuronoxylans. Alkaline delignification led to 90.3% lignin removal from steam-exploded materials, but its effect on enzymatic hydrolysis was almost negligible. For the best pretreatment condition, analyses by confocal laser scanning microscopy and solid-state nuclear magnetic resonance revealed important changes in fiber morphology and chemical composition, respectively. Enzymatic hydrolysis at 4 wt % total solids with Cellic CTec2 and Cellic CTec3 (Novozymes) led to 22.4 and 27.8 g L-1 glucose equivalents in 96 h, respectively, whose fermentation with a commercial strain of Saccharomyces cerevisiae led to ethanol productivities greater than 3.5 g L-1 in 6 h.