Although it carries two competent replication systems, a composite plasmid formed in vitro by linkage of the complete ColE1 and pSC101 plasmid replicons at their unique EcoRI endonuclease cleavage sites normally uses only the replication origin and functions of the ColE1 component. Restriction of ColE1 replication functions by DNA polymerase I deprivation results, however, in exclusive use of the pSC101 replication origin. When using the ColE1 replication system the composite plasmid is nevertheless incompatible with both the parent replicons. This suggests that a trans-dominant gene product is involved in plasmid incompatibility and supports negative control rather than positive control models for regulation of the initiation of DNA replication.