The gene encoding the principal Butyrivibrio fibrisolvens xylosidase (xylB) has been cloned and expressed in Escherichia coli under the control of the lac promoter. The coding region for this gene was localized within a 3.2-kilobase B. fibrisolvens DNA fragment in pUC18. A new protein band was observed in recombinant E. coli containing xylB. This protein (approximately 60,000 molecular weight) was presumed to be the xylosidase monomer. The optimal pH (5.5) and substrate range for the recombinant and native xylosidases appeared identical. Both enzymes hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 5 and both were inactive on xylan.