The biological control bacterium Pseudomonas aureofaciens 30-84 utilizes an N-acyl-homoserine lactone (AHL) signal molecule to control phenazine antibiotic production in the wheat rhizosphere (D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). In this study, we demonstrate that naturally co-existing, non-isogenic bacterial populations interact with strain 30-84 at the level of gene expression via the exchange of diffusible signals on wheat roots. Wheat plants from three geographic locations were used to generate a random library of 700 rhizobacterial isolates. Roughly 8% of the isolates from each location restored phenazine gene expression to an AHL-deficient strain of 30-84 in vitro. Five of these isolates were further tested for their ability to influence gene expression of an AHL-deficient reporter of strain 30-84 on wheat roots. All five, isolated from different geographic locations, restored phenazine gene expression by the reporter to wild-type levels. This suggests that in vitro assays can identify bacterial isolates with the potential to influence phenazine expression in strain 30-84 via AHLs on wheat roots. The occurrence of such strains in all fields sampled suggests that AHL-mediated communication is a common occurrence in the wheat rhizosphere.