TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein shows in vitro DNA helicase activity. Here we report that it also has in vitro oriT-endonuclease activity. TrwC specifically nicks oriT-containing supercoiled plasmid DNA in the presence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phosphorylation by T4 polynucleotyde kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions in oriT indicated that the nucleotide sequences immediately surrounding the nic site are important, but not the only essential feature, for the nicking reaction. Comparison of the R388 nic site with previously published nic DNA sequences suggests that IncF, IncN and IncW plasmids form a family of related nic sites. During the course of this work we have also demonstrated a terminal transferase activity of Sequenase Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mapped nic sites in other systems.