Although the m-xylene-responsive sigma54 promoter Pu of Pseudomonas putida mt-2, borne by the TOL plasmid pWWO, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. This is unusual because regulatory networks (such as the one to which Pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. This study provides genetic evidence that the upstream-activating sequences (UAS), which serve as the binding sites for the pWW0-encoded XylR protein (the m-xylene-responsive sigma54-dependent activator of Pu), isolate expression of the upper TOL genes from any adventitious transcriptional flow originating further upstream. An in vivo test system was developed in which different segments of the Pu promoter were examined for the inhibition of incoming transcription products from an upstream promoter in P. putida and Escherichia coli. Minimal transcription filter ability was located within a 105-bp fragment encompassing the UAS of Pu. Although S1 nuclease assays showed that the UAS prevented the buildup of downstream transcripts, the mechanism seems to diverge from a typical termination system. This was shown by the fact that the UAS did not halt transcription in vitro and that the filter effect could not be relieved by the anti-termination system of lambda phage. Because the Pu promoter lies adjacent to the edge of a transposon in pWW0, the preset transcriptional filter in the UAS may isolate the upper TOL operon from undue expression after random insertion of the mobile genetic element in a new replicon.