In order to elucidate the molecular mechanism of plasmid incompatibility of broad host-range plasmid 81162, the plasmid-encoded replication protein RepIB was purified and tested for binding to the 20 by direct repeat (DR) DNA sequence which is reiterated 3 and 1/2 times within the replicative origin of the plasmid. The RepIB protein specifically binds to the DR DNA. Point mutations in the DR which affect expression of plasmid incompatibility also coordinately affect binding. These results indicate that the incompatibility of broad host-range plasmid 81162 is exerted by the DR DNA by titrating the essential replication protein RepIB.