The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (PHL) is a major determinant in the biological control of a range of plant pathogens by many fluorescent Pseudomonas spp. A 4.8-kb chromosomal DNA region from Pseudomonas fluorescens Q2-87, carrying PHL biosynthetic genes, was used as a probe to determine if the PHL biosynthetic locus is conserved within PHL-producing Pseudomonas strains of worldwide origin. The phl gene probe hybridized with the genomic DNA of all 45 PHL-producing Pseudomonas strains tested, including well-characterized biocontrol strains from the United States and Europe and strains isolated from disease-suppressive soils from Switzerland, Washington, Italy, and Ghana. The PHL producers displayed considerable phenotypic and genotypic diversity. Two phenotypically distinct groups were detected. The first produced PHL, pyoluteorin, and hydrogen cyanide and consisted of 13 strains from almost all locations sampled in the United States, Europe, and Africa. The second produced only PHL and HCN and consisted of 32 strains from the U.S. and European soils. Analysis of restriction patterns of genomic DNA obtained after hybridization with the phl gene probe and cluster analysis of restriction patterns of amplified DNA coding for 16S rRNA (ARDRA) and randomly amplified polymorphic DNA (RAPD) markers indicated that the strains that produced both PHL and pyoluteorin were genetically highly similar. In contrast, there was more diversity at the genotypic level in the strains that produced PHL but not pyoluteorin. ARDRA analysis of these strains indicated two clusters which, on the basis of RAPD analysis, split into several subgroups with additional polymorphisms. In general, the occurrence of phenotypically and genotypically similar groups of PHL producers did not correlate with the geographic origin of the isolates, and highly similar strains could be isolated from diverse locations worldwide.