The tcp conjugation locus is present in both toxin and antibiotic resistance plasmids from the Gram-positive anaerobic human and animal pathogen Clostridium perfringens. To determine the functional role of the proteins encoded by this locus and to develop a model for the conjugation process we have mutated each of the 11 tcp genes located on our paradigm 47.2 kb tetracycline resistance plasmid pCW3 and shown that five of these genes are essential for conjugative transfer (tcpADEFH) and that three genes are essential for efficient transfer (tcpMCG). We have identified novel relaxase (TcpM) and coupling proteins (TcpA) and structural analysis of TcpC showed that it was structurally related to the VirB8 family. TcpC is now regarded as the prototype for the β-subclass of VirB8 proteins. We have shown that oriT is located upstream of the first gene in the tcp operon, tcpM, and have demonstrated that TcpM binds to the oriT site. We also have demonstrated that the conserved tcpK gene, which is located upstream of oriT and is therefore outside the original tcp locus, is also required for efficient transfer. TcpK appears to be part of the relaxosome complex since it causes a supershift of an oriT fragment in the presence of TcpM. Surface plasmon resonance studies have shown that TcpK binds to repeat sequences that form part of the oriT site and X-ray crystallographic analysis has indicated that TcpK is structurally related to DNA binding proteins from other bacteria. The studies have expanded our understanding of the functional biology of this unique family of conjugative plasmids.