Pseudomonas fluorescens strain CHAO is an effective biocontrol agent of diseases caused by soilborne plant pathogens. Strain CHAO produces several secondary metabolites, notably cyanide, acetylpheoroglucinols and pyoluteorin. Cyanide plays an important role in the suppression of black root rot of tobacco, caused by Thielaviopsis basicola. A mutant, CHA625, has now been isolated, which does not produce 2,4-diacetylphloroglucinol. Strain CHA625 suppressed black root rot to a distinctly smaller extent than did wild-type CHAO under gnotobiotic conditions. A cosmid obtained from a genomic library of strain CHAO restored the ability of strain CHA625 to produce this metabolite and to suppress disease. Addition of synthetic 2,4-diacetylphloroglucinol to soil reduced disease severity in the absence of bacteria. These results suggest that the production of 2,4-diacetylphloroglucinol by P. fluorescens strain CHAO is another important factor, in addition to cyanide, in the suppression of black root rot of tobacco.