An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase activity were pH 10 and 80 degrees C, respectively. This xylanase was stable within a pH range of 5-10 and up to a temperature of 90 degrees C. It showed high thermostability at 60 degrees C for 5 days and half-life times at 90 degrees C and 100 degrees C were 2 and 1 h, respectively. The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan. This enzyme obeyed the Michaelis-Menten kinetics, with the Km and k(cat) values being 1.55 mg soluble oat-spelt xylan/ml and 388 min(-1), respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn2+, Ca2+, and Cu2+, it was, strongly inhibited by Hg2+, Zn2+, and Ba2+. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.