The replication region of the 111-kb circular plasmid pKNR from Rhodococcus opacus B-4 was identified. A PCR-based deletion analysis using the λ Red recombination technique followed by restriction digestion and PCR-amplification analyses revealed that a 2.5-kb fragment covering one putative open reading frame (ORF) was involved in the replication of pKNR. The product of this ORF showed significant similarity to a functionally unknown protein encoded in the replication region of the 70-kb circular plasmid of Clavibacter michiganensis and to ones in other bacterial large circular plasmids. These observations suggest that the product of the identified ORF and its orthologs can serve as novel replication proteins for large circular bacterial plasmids.