In the worldwide quest for producing biofuels from lignocellulosic biomass, the importance of the substrate pretreatment is becoming increasingly apparent. This work examined the effects of reducing the substrate particle sizes of wheat straw by grinding prior to wet oxidation and enzymatic hydrolysis. The yields of glucose and xylose were assessed after treatments with a benchmark cellulase system consisting of Celluclast 1.5 L (Trichoderma reesei) and Novozym 188 beta-glucosidase (Aspergillus niger). Both wet oxidized and not wet oxidized wheat straw particles gave increased glucose release with reduced particle size. After wet oxidation, the glucose release from the smallest particles (53-149 mu m) reached 90% of the theoretical maximum after 24 h of enzyme treatment. The corresponding glucose release from the wet oxidized reference samples (2-4 cm) was similar to 65% of the theoretical maximum. The xylose release only increased (by tip to 39%) with particle size decrease for the straw particles that had not been wet oxidized. Wet oxidation pretreatment increased the enzymatic xylose release by 5.4 times and the glucose release by 1.8 times across all particle sizes. Comparison of scanning electron microscopy images of the straw particles revealed edged, nonspherical, porous particles with variable surface structures as a result of the grinding. Wet oxidation pretreatment tore lip the surface structures of the particles to retain vascular bundles of xylem and phloem. The enzymatic hydrolysis left behind a significant amount of solid, apparently porous structures within all particles size groups of both the not wet oxidized and wet oxidized particles. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 399-408, 2009