The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD(2)C(2). To assess the extent to which FlhD(2)C(2) directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42-44 bp inverted repeat FlhD(2)C(2) binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD(2)C(2) binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD(2)C(2) over the K(D) range 38-356 nM, and of the nine corresponding chromosomal promoter-lacZ fusions, those of the four genes b1904, b2446, wzz(fepE) and gltI showed up to 50-fold dependence on FlhD(2)C(2) in vivo. In comparison, four representative flagella class II promoters bound FlhD(2)C(2) in the K(D) range 12-43 nM and were upregulated in vivo 30- to 990-fold. The FlhD(2)C(2)-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted -35 motif of the FlhD(2)C(2)-activated sigma(70) promoters, as is the case with FlhD(2)C(2)-dependent class II flagellar promoters. The data indicate a wider FlhD(2)C(2) regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.