In one family of bacterial plasmids, multiple initiator binding sites, called iterons, are used for initiation of plasmid replication as well as for the control of plasmid copy number. Iterons can also pair in vitro via the bound initiators. This pairing, called handcuffing, has been suggested to cause steric hindrance to initiation and thereby control the copy number. To test this hypothesis, we have compared copy numbers of isogenic miniP1 plasmid monomer and dimer. The dimer copy number was only one-quarter that of the monomer, suggesting that the higher local concentration of origins in the dimer facilitated their pairing. Physical evidence consistent with iteron-mediated pairing of origins preferentially in the dimer was obtained in vivo. Thus, origin handcuffing can be a mechanism to control P1 plasmid replication.