The Sym plasmid pRL1JI encodes functions for the formation of nitrogen-fixing pea root nodules by Rhizobium leguminosarum. Some of the nodulation genes are involved in recognition of chemical signals produced by the plant root, and others are required for production of chemical signals recognized by the plant. pRL1JI also contains a regulatory gene, rhiR, that is homologous to luxR, the transcriptional activator of luminescence genes in Vibrio fischeri. LuxR requires a signal compound, an autoinducer, for its activity. We have identified an R. leguminosarum autoinducer that, together with RhiR, is required to activate both the rhizosphere-expressed rhiABC operon and a growth-inhibiting function encoded by pRL1JI. This intercellular signal is an N-acylated homoserine lactone structurally related to the V. fischeri and other autoinducers. These findings indicate a new level of intercellular communication in root nodule formation.