A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 bp synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.