In many bacteria, gene replacement can only be achieved using RP4-based conjugative systems where exogenous DNA is delivered to the recipient on conditionally replicating plasmids carrying an oriT(RP4). In the commonly used strains SM10 and S17-1 conjugative functions are provided by an RP4 plasmid inserted in the chromosome. Those strains, besides mobilizing oriT(RP4)-carrying plasmids, also transfer their own chromosomal genes, which can be inconvenient for several reasons. We describe here a new version of an RP4-based conjugative system that allows for efficient transfer of mobilizable plasmids without the transfer of chromosomal genes, providing fully controlled and efficient exogeneous DNA delivery to non-transformable bacteria.