The ColE2-P9 rep protein specifically binds to the orgin and initiates DNA synthesis. Interaction of the Rep protein with the origins of plasmids ColE2-P9 and ColE-3-CA38 (one of the close relatives of ColE2-P9) is plasmid-specific. By using chimeric rep genes and chimeric origins we showed that the two region, A and B, in the C-terminal regions of the Rep proteins and the two sites alpha and beta, in the origins are important for the determination of specificity. When each of the A/alpha and B/beta pairs is from the same plasmids, the plasmid replication is efficient. On the other hand, if only the A/alpha pair is from the same plasmids, the plasmid replication is inefficient. For the region A, the plasmid-specificity is mainly determined by the presence or absence of a nine-amino acid sequence. For the region B, the specificity is probably determined by several amino acids. The region B, contains a segment of amino acid sequence which shows significant homology with the DNA recognition helices of various DNA binding proteins. At the site alpha, the single additional base-pair in the ColE3-CA38 origin can be either A/T or T/A. At the site beta, however, the single additional base-pair in the ColE2-P9 origin must be G/C. Among other possibilities we propose that the region A is a linker connecting the two domains in the Rep protein involved in DNA-binding and that the region B is a part of the sequence-specific DNA-binding domain.