The gene encoding xylanase activity in the ruminal bacterium Bacteroides ruminicola D31d was cloned and expressed in Escherichia coli with the plasmid vector pUC18. The gene was isolated on a 4.7-kilobase pair partial PstI genomic DNA fragment. The xylanase activity expressed in E. coli was cell associated and could degrade both oatspelt xylan and Remazol Brilliant Blue-xylan. The xylanase did not have detectable activity against carboxymethylcellulose. Utilization of an endogenous promoter by E. coli was indicated by expression of xylanase activity after subcloning of the insert into pBR322 in opposite orientations. The B. ruminicola D31d xylanase gene was compared by Southern hybridization analyses with xylanase genes cloned from B. ruminocola 23 and B. ovatus V975, a human intestinal isolate. The D31d xylanase gene did not cross-hybridize with either of the other two genes. In addition, the 23 xylanase gene did not cross-hybridize with the other two genes according to the same technique. These results indicate that the three cloned genes do not share a high degree of genetic similarity, despite the similar enzymatic activities. This is the first study to compare cloned genes from ruminal and colonic Bacteroides species.