Tumour-inducing (Ti) plasmids of Agrobacterium tumefaciens replicate via the products of the repABC genes, which are highly conserved among plasmids of the alpha-Proteobacteria. RepA and RepB direct stable partitioning of daughter plasmids, while the RepC directs replicative DNA synthesis. We have identified a new gene (repE) within the repB-repC intergenic region of an octopine-type Ti plasmid. This gene encodes a small, non-translated RNA that is transcribed in the direction opposite to the repABC mRNA. Increased expression of repE blocked plasmid replication of a repABC-dependent miniplasmid, while decreased repE expression increased plasmid copy number. Direct RNA measurements and repC-lacZ fusions demonstrated that RepE inhibits the expression of RepC at the transcriptional level and possibly also at the translational level. Based on our experimental results and an RNA folding algorithm, we predict that RepE binding to the repABC mRNA would promote termination of the repABC transcript near the start codon of repC. Sequence analysis suggests that this phenomenon may be widespread among plasmids of this family.