The xylanase gene cluster from the rumen anaerobe Prevotella bryantii B(I)4 was found to include a gene (xynR) that encodes a multidomain regulatory protein and is downstream from the xylanase and P-xylosidase genes xynA and xynB. Additional genes identified upstream of xynA and xynB include xynD, which encodes an integral membrane protein that has homology with Na:solute symporters; xynE, which is related to the genes encoding acylhydrolases and arylesterases; and xynF, which has homology with the genes encoding alpha-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a xynA probe, and the start site of the 3.7-kb transcript encoding xynABD was mapped to a position upstream of xynD. The DNA binding domain of XynR was purified after amplification and overexpression in Escherichia coli and was found to bind to a 141-bp DNA fragment from the region immediately upstream of xynD. In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of xynABD in P. bryantii B(I)4. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.