Replication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small oriRK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus ‘incompatible” with RK2 replicons. However, the 393 bp oriRK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on oriRK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII oriRK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from oriRK2is required or may simply reflect the strong selective pressure for establishment of the incoming oriRK2 plasmid and poor ability of the HpaII oriRK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII oriRK2 may be designated inc1. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc2 region, to distinguish it from inc1, above, that overlaps but does not coincide with the 393 bp HpaII oriRK2. This inc2 region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete oriRK2. Also, only part of the inc2 region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for oriRK2.