Functional genetic analysis of the novel tcp locus has revealed that it mediates the conjugative transfer of the toxin and antibiotic resistance plasmids from Clostridium perfringens. This locus has nine conserved genes that are required for efficient conjugative transfer of the paradigm tetracycline resistance plasmid, pCW3. We have now examined the role of the unique membrane proteins encoded by the tcpD and tcpE genes and have shown that they are essential for conjugation. Localisation studies using functional HA-tagged TcpD and TcpE derivatives in C. perfringens showed that TcpD and TcpE localised to the cell envelope, mostly at the poles of donor cells, suggesting that they form part of the transfer apparatus. Surprisingly, neither a relaxase nor an origin of transfer (oriT) has been identified on pCW3. The first gene of the tcp locus, intP, encodes a putative tyrosine recombinase. An intP mutant was shown to have a significantly lower conjugation frequency and complementation with the wild-type intP gene in trans restored transfer to wild-type levels. An intP-Y259F derivative was unable to restore transfer, demonstrating that the Y259 residue was essential for IntP function, which was consistent with the need for a hydrophilic attack at the oriT site. In addition, a putative accessory protein, TcpK, was identified as a potential component of the pCW3 relaxosome and shown to be required for wild-type pCW3 transfer. Finally, mobilisation studies defined the pCW3 oriT site as a 148 nt region upstream of the intP gene. Gel shift assays showed that IntP bound specifically to the oriT site and that a supershift was observed in the presence of both IntP and TcpK. In conclusion, we postulate that IntP is an atypical relaxase that together with TcpK initiates conjugative transfer by binding to and processing the oriT site.