Pseudomonas fluorescens strain CHA0 suppresses Thielaviopsis basicola-induced black root rot of tobacco and Gaeumannomyces graminis var. tritici-induced take-all of wheat. Strain CHA0 produces 2,4-diacetylphloroglucinol, a metabolite with antifungal, antibacterial, and phytotoxic activity. The role of this compound in disease suppression was tested under gnotobiotic conditions. A P. fluorescens mutant, obtained by Tn5 insertion, did not produce 2,4-diacetylphloroglucinol, showed diminished inhibition of T. basicola and of G. g. var. tritici in vitro, and had a reduced suppressive effect on tobacco black root rot and on take-all of wheat, compared with wild-type CHA0. Complementation of the mutant with an 11-kb DNA fragment from a genomic library of wild-type CHA0 largely restored production of the metabolite, inhibition of the fungal pathogens in vitro and disease suppression. The Tn5 insertion was physically mapped using a 5.8-kb complementing fragment as a probe. 2,4-Diacetylphloroglucinol was shown to be produced in the rhizosphere of wheat by strain CHA0 and by the complemented mutant, but not by the mutant defective in 2,4-diacetylphloroglucinol synthesis. These results support the importance of 2,4-diacetylphloroglucinol production by strain CHA0 in the suppression of soilborne plant pathogens in the rhizosphere.