Aspergillus niger IFO 4066 produced two xylanases, xylanase A (XynNA) and xylanase B (XynNB), in culture medium, and these enzymes were purified. Acidophilic xylanase such as xylanase C (XynC) of white koji mold (Aspergillus kawachii IFO 4308) was not detected in A. niger cultures. However, results of Southern analysis using xynC cDNA of A. kawachii as a probe suggested that A. niger contained a gene homologous to xynC of A. kawachii. Therefore, we cloned this xylanase gene from A. niger. The predicted amino acid sequence of the cloned xylanase showed a homology to that of xynC of A. kawachii. However, a large number of amino acid substitutions were detected, especially in the N-terminal region. Both this cloned gene and xynC gene of A. kawachii had an intron at the same position in the coding region. The cloned gene was expressed in A. kawachii and a large quantity of xylanase was produced. The elution profile on an anion exchange chromatogram and the N-terminal amino acid sequence of the xylanase purified from the transformant were the same as those of XynNB. This confirmed that the cloned gene encoded XynNB.