For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity. The trfA gene is transcribed as part of a polycistronic operon which also includes kilD. Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci. Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself. Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter. This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans. When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2. Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis. These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication.