Expression of the tumor-inducing (Ti) plasmid virulence genes of Agrobacterium tumefaciens is required for the transfer of DNA from the bacterium into plant cells, ultimately resulting in the initiation of plant tumors. The vir genes are induced as a result of exposure to certain phenol derivatives, monosaccharides, and low pH in the extracellular milieu. The soil, as well as wound sites on a plant-the usual site of the virulence activity of this bacterium-can contain these signals, but vir gene expression in the soil would be a wasteful utilization of energy. This suggests that mechanisms may exist to ensure that vir gene expression occurs only at the higher concentrations of inducers typically found at a plant wound site. In a search for transposon-mediated mutations that affect sensitivity for the virulence gene-inducing activity of the phenol, 3,5-dimethoxy-4-hydroxyacetophenone (acetosyringone [AS]), an RND-type efflux pump homologous to the MexE/MexF/OprN pump of Pseudomonas aeruginosa was identified. Phenotypes of mutants carrying an insertion or deletion of pump components included hypersensitivity to the vir-inducing effects of AS, hypervirulence in the tobacco leaf explant virulence assay, and hypersensitivity to the toxic effects of chloramphenicol. Furthermore, the methoxy substituents on the phenol ring of AS appear to be critical for recognition as a pump substrate. These results support the hypothesis that the regulation of virulence gene expression is integrated with cellular activities that elevate the level of plant-derived inducers required for induction so that this occurs preferentially, if not exclusively, in a plant environment.IMPORTANCE Expression of genes controlling the virulence activities of a bacterial pathogen is expected to occur preferentially at host sites vulnerable to that pathogen. Host-derived molecules that induce such activities in the plant pathogen Agrobacterium tumefaciens are found in the soil, as well as in the plant. Here, we tested the hypothesis that mechanisms exist to suppress the sensitivity of Agrobacterium species to a virulence gene-inducing molecule by selecting for mutant bacteria that are hypersensitive to its inducing activity. The mutant genes identified encode an efflux pump whose proposed activity increases the concentration of the inducer necessary for vir gene expression; this pump is also involved in antibiotic resistance, demonstrating a relationship between cellular defense activities and the control of virulence in Agrobacterium.