Transfer of the Rhizobium leguminosarum biovar viciae symbiosis plasmid pRL1JI is regulated by a cascade of gene induction involving three LuxR-type quorum-sensing regulators, TraR, BisR and CinR. TraR induces the plasmid transfer traI-trb operon in a population-density-dependent manner in response to N-acylhomoserine lactones (AHLs) made by TraI. Expression of the traR gene is primarily induced by BisR in response to AHLs made by CinI, and expression of cinI is induced by CinR and repressed by BisR. Analysis of transcription initiation of cinI, traR and traI identified potential regulatory domains recognized by the CinR, BisR and TraR regulators. Deletion and mutation of the cinI promoter identified potential recognition motifs for activation by CinR and repression by BisR. Analysis of the DNA sequence upstream of traI and expression of transcriptional gene fusions revealed a predicted TraR-binding (tra-box) domain. Two transcript initiation sites were identified upstream of the plasmid replication gene repA, which is divergently transcribed from traI; one of these repA transcripts requires the quorum-sensing cascade mediated via BisR and TraR, showing that the pRL1JI plasmid replication genes are co-regulated with the plasmid transfer genes.