The novel tcp conjugation locus mediates conjugative transfer of the unique toxin and resistance plasmids from Clostridium perfringens. Previous studies have identified several tcp-encoded proteins that form the transfer apparatus. Surprisingly, neither a relaxase nor an origin of transfer (oriT) have been identified, although they are essential for plasmid processing prior to transfer. The first gene of the tcp locus, intP, encodes a potential tyrosine recombinase. An intP mutant of the tetracycline resistance plasmid pCW3 was constructed and shown to have a significantly lower conjugation frequency compared to wild type. Complementation with the wild-type intP gene in trans restored transfer to wild-type levels. However, an IntP-Y259F derivative was unable to restore transfer, demonstrating that the Y259 residue was essential for IntP function and suggesting that the hydroxyl group was necessary for IntP catalytic activity, which is consistent with the need for a hydrophilic attack at the oriT site. Additional studies showed that other conserved tyrosine recombinase residues were not required for IntP activity. A putative plasmid encoded helicase, TcpK, was identified as a potential component of the relaxosome. Mutagenesis of the tcpK gene led to a significant reduction in conjugation frequency, which returned to wild-type levels upon complementation. Finally, a 391-bp intergenic region upstream of the intP gene was predicted to encode the oriT site. Mobilisation studies showed that the presence of this region significantly increased the mobilisation frequency of a shuttle vector. Further studies defined the minimal pCW3 oriT site as a 148 bp fragment of this intergenic region and gel mobility shift assays showed that IntP bound specifically to the oriT site. In conclusion, we postulate that IntP is a novel relaxase that together with TcpK is involved in the initiation of conjugative transfer by binding to and processing the oriT site located proximal to the tcp locus.