Plasmid pRO101, a derivative of plasmid pJP4 which contains Tn1721 inserted into a nonessential region, is inducible for 2,4-dichlorophenol hydroxylase (DCPH) encoded by tfdB. Plasmid pRO103, which has a deletion in the BamHI-F-BamHI-E region of plasmid pRO101, has elevated basal levels of DCPH but is uninducible. The regulatory gene for tfdB, designated tfdS, was cloned as an 8.3-kilobase-pair EcoRI-E fragment. When the cloned tfdS gene was in trans with plasmid pRO103, the baseline DCPH levels were repressed to normal uninduced levels and were fully induced when this strain was grown in the presence of 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol, or 4-chlorocatechol. However, when tfdS was in trans with tfdB in the absence of tfdCDEF, tfdB was repressed but could not be induced. When tfdS and tfdC1, which encodes chlorocatechol 1,2-dioxygenase, are in trans with tfdB, tfdB remained uninduced, indicating that a downstream metabolite of chloro-cis,cis-muconate, either 2-cis-chlorodiene lactone or chloromaleylacetic acid, is the effector. Collectively, these data demonstrate that the gene product of tfdS acts as a repressor of tfdB in the absence of an effector and as an activator of tfdB when an effector is present. (Copyright (c) 1990 American Society for Microbiology.)