The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters P-A and P-F, respectively. However, there is an additional weak promoter, P-R, which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the P-A and P-F promoters were activated by D-xylose and repressed by glucose. In contrast, the P-R promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the P-A and P-F promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was positively regulated by XylR. In vivo footprinting analysis revealed that XylR binds to at least two DNA regions, I-A and I-F, each with a direct repeat. It is very likely that XylR interacts with I-A and I-F as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while I-A is additionally flanked by a cyclic AMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions.