Aims: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543. Methods and Results: The xylanase gene was cloned into pUC19 in Escherichia coli DH5 alphaF' and selected by growth on RBB-xylan. All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping. The nucleotide sequence of the P. stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da. The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites. The K-m values for non-glycosylated and glycosylated xylanases were 1.4 mg ml(-1) and 42 mg ml(-1), respectively, and V-max values were 0.8 and 0.082 mu mol min(-1) mg(-1) protein, respectively. Conclusions: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail. Significance and Impact of the Study: The results of this study can be used to develop better xylanase-utilizing yeast strains.