A thermostable extracellular xylanase was purified and characterized from brown-rot basidiomycete Laetiporus sulphureus, cultivated on biologically pretreated Pinus densiflora biomass. After three consecutive purification steps using DEAE, Mono Q, and Superdex 75 columns, the xylanase specific activity was found to be 72.4 U/mg, nine fold higher than that of the crude culture solution, purity was 96%. and the molecular mass determined to be 69.3 kDa. The optimal pH and temperature for xylanase activity were 3.0 and 80 degrees C, respectively. Although activity of xylanase was highest at 80 degrees C, it showed highest thermostability at 60 degrees C, retaining approximately 97% of its relative activity following incubation for 4 h. In the presence of 5 mM solution of CaCl2, the relative xylanase activity increased by 35.9%; however, it decreased significantly in the presence of 10 MM Solution of Cu2+. Among the xylan-based substrates tested, purified L sulphureus xylanase showed the highest activity on beechwood xylan. Thin-layer chromatography (TLC) experiments revealed that purified L sulphureus xylanase is an endoxylanase that hydrolyzes xylotriose, xylotetraose, and xylopentaose but not xylobiose. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.