Pseudomonas fluorescens Migula strain 3551, isolated from cotton rhizosphere soil, protected cotton from seed colonization and preemergence damping-off caused by Pythium ultimum. The role of fluorescent siderophore production by strain 3551 in its antagonism against P. ultimum was investigated. Fourteen nonfluorescent (Flu–) Tn5 insertion mutants of P. fluorescens 3551 were obtained following matings with Escherichia coli SM10 (pSUP1011). Strain 3551 grew on an iron-deficient medium, whereas the 14 Flu– derivative strains did not. Southern analysis of EcoRI digested genomic DNA from the Flu– mutants probed with nick-translated λ:Tn5 confirmed the presence of single insertions of Tn5 in the chromosomal DNA of each derivative strain. Representative mutants from five of nine EcoRI fragment size classes were compared to the parental strain with respect to antagonism against P. ultimum in the cotton spermosphere. Cotton seed treated with metalaxyl, strain 3551, or Flu– derivative strains were evaluated for colonization by P. ultimum 24 hr after planting into infested soil and for their subsequent emergence. Although the parental strain 3551 decreased colonization of cotton seed by P. ultimum and increased seedling emergence to a level statistically equivalent to metalaxyl treatment, Flu– derivative strains did not. Differences between 3551 and Flu– derivative strains were greater with respect to emergence than P. ultimum colonization. No consistent differences were observed between strain 3551 and Flu– derivative strains with respect to their growth rates or population sizes in the cotton spermosphere. Results indicate that fluorescent siderophore production by P. fluorescens 3551 contributes to but does not account for all of its antagonistic activity against P. ultimum.