This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.