Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLPproducing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.Cyclic lipopeptides (CLPs) are produced by distinctively different groups of bacteria, both gram-positive (20) and gramnegative (28). The high diversity of CLP-producing microorganisms (28) and differences in chemical structure suggest that the CLP compounds may serve different, and possibly multiple, purposes. This may explain why the specific role of CLP production is often unclear (28,40). For a limited number of CLPs (28), the reported functions include promotion of bacterial swarming (12, 26) and biosurfactant properties (19,24,41). In many cases, CLP compounds are also known to exert a role in antagonistic interactions with other organisms (28), e.g., plant pathogenicity (5) and antifungal (19,30,31,38,44), antibacterial (11), antiviral (49), or cytotoxic (16) activity.Synthesis of CLPs is nonribosomal and catalyzed by large peptide synthetase complexes (27). Various environmental stimuli may affect CLP production, i.e., carbon substrate (36), limitation by C, N, or P (15, 37), Fe limitation (15), growth phase conditions (15), and interaction with interfaces (32). Little information is available on production rates and regulating factors for the compounds in natural environments. Asaka and Shoda (2) detected surfactin and iturine production by Bacillus subtilis RB14 in a sterilized vermiculite-soil system, and Nakayama et al. (31) detected xanthobaccin A production by a Stenotrophomonas sp. strain, SB-K88, in a hydroponic sugar beet rhizosphere system, but documentation for in situ production of CLPs...