Pseudomonas fluorescens B16 is a plant glowth-prornoting rhizobacterium, which produces an antibacterial compound that is effective against plant root pathogens, such as Agrobacrerium tumefaciens and Raistonia solanacearum. We mutagenized the strain B16 with Omegon-Km and isolated six antibacterial-activity-deficient mutants. Two cosmid clones that hybridized with the mutant clones also were isolated from a genomic library of tile parent strain. Using deletion and complementation analyses, it was found that the biosynthesis genes resided in a 4.3-kb SalI-NarI fragment. When a plasmid clone carrying the fragment was introduced into P. fluorescens strain 1855.344, which does not exhibit any antibacterial activity, the transconjugants exhibited antibacterial activity, indicating that the plasmid clone carried all the genes essential for production of the antibacterial compound. DNA sequence analysis of the fragment identified four putative open reading frames (ORFs): orf1 through orf4 The deduced amino acid sequences of ORF1, ORF2, and ORF4 were similar to cystathionine gamma lyase, pyruvate formate-lyase activating enzyme, and transcriptional regulator, respectively, yet the amino acid sequence of ORF3 showed no similarities to any known proteins. It was also demonstrated that the antibacterial activity was responsible for biological control of the bacterial wilt caused by R. solanacearum.