Strain CHA0 of Pseudomonas fluorescens suppressed black root rot of tobacco, caused by Thielaviopsis basicola, under gnotobiotic conditions in an artificial soil containing vermiculite as clay mineral. When vermiculite was replaced by illite, P. fluorescens provided poor protection. Better protection was obtained by the addition of FeCl3 to illite. When Fe3+ chelated with ethylenediaminedi (o-hydroxyphenylacetic acid) (FeEDDHA) was added to illite, strain CHA0 gave good protection. Strain CHA400, a nonfluorescent, pyoverdine-negative mutant obtained by transposon insertion, protected the plant to the same extent as did the wild-type strain CHA0 in the systems containing vermiculite, illite, or illite amended with FeCl3. However, in the system containing illite and FeEDDHA, strain CHA400 gave poor protection, in contrast to strain CHA0. No differences were observed between CHA400 and CHA0 with respect to the intensity of root colonization. The survival of T. basicola was similar in both the vermiculite and the illite systems. The availability of iron in vermiculite clay was higher than in illite, as indicated by the amount of iron in soil water solution and the amount of HCN produced by P. fluorescens in minimal medium amended with the clay minerals. From these experiments, it appears that the suppression of black root rot by strain CHA0 requires sufficient iron, and iron competition is not a suppressive mechanism in this system. We hypothesize that HCN production has a role in disease suppression.