An extracellular endo-1,4-beta-xylanase was purified from the culture filtrate of a filamentous fungus, Penicillium citrinum strain FERM P-15944, grown on birch-wood xylan. The purified enzyme showed a single band on SDS-PAGE with an apparent M-r of 31.6 kDa. The xylanase activity was optimal at pH 6.0 and 50 degrees C. Southern blot analysis indicated that the xylanase gene (xynB) was present as a single copy in the genome. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. An open reading frame of xynB was interrupted by nine introns and encoded a presumed prepropeptide of 25 amino acids and a mature protein of 302 amino acids. Sequence alignment and the constructed neighbor-joining tree showed that the P. citrinum enzyme belongs to glycoside hydrolase family 10 and is closely related to several other xylanases from penicillia and black aspergilli. The xynB cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris.