An extracellular endo-1,4-beta-xylanase was purified from the culture filtrate of a filamentous fungus Penicillium citrinum FERM P-15944 grown on birch-wood xylan. The purified enzyme showed a single band on SDS-PAGE with an apparent M-r of 20,000 and had an isoelectric point below 3.5. Xylanase activity was optimal at pH 5.0 and 55 degrees C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. Southern blot analysis indicated that the xylanase gene (xynA) was present as a single copy in the genome. An open reading frame of 657 bp was interrupted by two introns of 65 and 55 bp, and encoded a presumed prepropeptide of 27 amino acids and a mature protein of 190 amino acids. Three distinct transcription start points were observed at positions -20 (A), -31 (A), and -36 (A) from the start codon. The 5'-noncoding region had a putative TATA box at nt-66 (TATAAA). The xynA cDNA was functionally expressed under the control of the alcohol oxidase I gene promoter in the methylotrophic yeast Pichia pastoris. A neighbor-joining tree showed that the P citrinum enzyme is closely related to several other fungal xylanases belonging to the glycoside hydrolase family 11: Trichoderma reesei XYN2, Aspergillus niger xynNB, Penicillium funiculosum xynC, Penicillium sp. strain 40 xynA, Chaetomium gracile cgxB, and Aspergillus nidulans xlnA and xlnB.