A β-xylosidase gene from Bacillus subtilis was cloned via the expression vector pKK223-3 into E. coli. The resulting recombinant strain E. coli [pKK223-300] produced a xylosidase which was cell-wall bound. The enzyme was dissociated from the cell wall with CHAPS detergent, and purified by affinity chromatography. The active form of the enzyme was a tetramer with subunit molecular weight 65, 000. p-Nitrophenyl β- xyloside and xylobiose were substrates while other glycosides and cellobiose were not. The enzyme was most active at 50°C and pH 7. Activity was enhanced at high sodium chloride concentration and inhibited by xylose. A unique amino-terminal amino acid sequence (30 residues) was found which indicates that the subunits are identical. The amino acid composition differed from that of a B. pumilus β-xylosidase determined previously (CLAEYSSENS et al., Biochim. Biophys. Acta, 405, 475 (1975)). The two enzymes also differed in cell location, subunit activity, and inhibition characteristics.