Plasmid R6K contains two functional origins or transfer (oriT), in contrast to previously characterized conjugative plasmids. The oriTs are formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the alpha and beta origins of replication. The gene for R6K oriT-nickase, taxC, was identified by transposon mutagenesis and sequenced, revealing that TaxC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stranded DNA containing its target sequence. Identification of the nic sites suggested that the R6K oriTs belong to the RP4/VirD2 oriT family. Cleavage was highly specific and did not occur with oligonucleotides cleaved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid. nic cleavage of in vivo preassembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphodiesterase to produce double strand cleavages at the nic sites. 35% of the molecules were cleaved simultaneously at both nic sites, both in the case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum percentage of individual R6K molecules containing two pre-assembled relaxation complexes.