We have developed a simple and efficient method of vector-mediated excision (VEX) for in vivo generation of precisely defined deletions in large bacterial genomes. The system uses homologous recombination with small cloned fragments on specialized pVEX plasmids to insert directly repeated bacteriophage P1 loxP sites at positions flanking the region to be deleted. In the presence of Cre recombinase, the loxP sites are efficiently recombined to produce the deletion. Deletion endpoints can be directed to specific nucleotides because they are defined by the termini of small homology-bearing fragments cloned into the pVEX plasmids. We have used VEX to delete trfA, the only known replication initiator gene of the 56.4 kb broad host-range plasmid RK2. The RK2 delta trfA mutant was found to be conjugation proficient, but unable to replicate in the RK2 hosts Acinetobacter calcoaceticus, Caulobacter crescentus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Rhizobium meliloti, or Rhodobacter sphaeroides. These results show that trfA is essential for replication in these hosts and indicate that the broad host-range of RK2 does not involve multiple replicons.