The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected. The requirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can be recovered as a plasmid only if RepA is provided in trans. These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.