We examined the genetic basis and transfer for production of rhizobitoxine, an inhibitor of ethylene biosynthesis in plants, directed by the rtx genes of Bradyrhizobium elkanii. Comparison with genome sequences of Bradyrhizobium japonicum and Xanthomonas oryzae suggests that the rtx genes extend from the previously identified rtxAC genes through four additional genes rtxDEFG. Reverse transcription-PCR analysis showed that the rtxACDEFG genes are expressed as an operon. Mutational analysis indicated that rtxDEG mutants reduced rhizobitoxine biosynthesis, while the rtxA gene is essential for its synthesis. Introduction of the rtxACDEFG into Agrobacterium tumefaciens resulted in strong expression of rtxACDEFG and production of RtxA protein, but no rhizobitoxine was detectable. Addition of O-acetylhomoserine, a precursor of rhizobitoxine, to the Agrobacterium derivative, however, fostered production of rhizobitoxine in culture. The diluted culture supernatant inhibited the activities of beta-cystathionase and 1-aminocyclopropane-1-carboxylate synthase, indicating that A. tumefaciens carrying rtxACDEFG genes excreted biologically active rhizobitoxine.