The hpa cluster of Escherichia coli W ATCC 11105 encodes the enzymes involved in the catabolism of 4-hydroxyphenylacetate (4-HPA). The catabolic genes are organized in two operons, the hpaBC operon, which produces the hydroxylase activity, and the meta operon, which encodes the enzymes that cleave the aromatic ring and allows its further metabolism. Using a monocopy or multicopy lacZ reporter system, we have demonstrated that the hydroxylase genes are transcribed from the PBC promoter which is positively regulated by the hpaA gene product. HpaA is activated by 4-HPA, 3-HPA, or phenylacetate and represents a novel member of the AraC/XylS family of regulators that recognizes aromatic effectors. The -35 box of the PBC promoter clearly deviates from the consensus sigma 70 promoters of E. coli, but upstream of this box we observed two direct repeats, a common characteristic of promoters regulated by the AraC family of proteins. The hpaA gene, which appears to form a transcriptional unit with the putative hpaX transport gene, is also expressed from an alternative promoter that is located within the hpaX structural sequence. On the basis of these findings, we propose a working model for the regulation of the unique aromatic catabolic pathway thus far characterized at a molecular level in E. coli. This regulatory circuit opens a new scenario for the development of novel microbiological tools for environmental purposes.