We have isolated plasmids by linking the 5.9 MD EcoRI fragment of E. coli that carries the origin of replication to an EcoRI fragment that carries an amplicillin resistance determinant, but lacks an origin of replication. 3 plasmids of this type, pOC1, pOC2, and pOC3, are described in detail in this report. Although the plasmids have some adverse effect on the growth properties of the host strain, their existence shows that two functioning chromosomal origins can coexist in one cell. Deletions generated from this type of plasmids allow an allocation of the origin of replication of E. coli within a DNA segment less than 0.4 MD in size.