Bacterial conjugation is important for the acquisition of virulence and antibiotic resistance genes. We investigated the mechanism of conjugation in Gram-positive pathogens using a model plasmid pCW3 from Clostridium perfringens. pCW3 encodes tetracycline resistance and contains the tcp locus, which is essential for conjugation. We showed that the unique TcpC protein (359 amino acids, 41 kDa) was required for efficient conjugative transfer, localized to the cell membrane independently of other conjugation proteins, and that membrane localization was important for its function, oligomerization and interaction with the conjugation proteins TcpA, TcpH and TcpG. The crystal structure of the C-terminal component of TcpC (TcpC(99-359)) was determined to 1.8-A resolution. TcpC(99-359) contained two NTF2-like domains separated by a short linker. Unexpectedly, comparative structural analysis showed that each of these domains was structurally homologous to the periplasmic region of VirB8, a component of the type IV secretion system from Agrobacterium tumefaciens. Bacterial two-hybrid studies revealed that the C-terminal domain was critical for interactions with other conjugation proteins. The N-terminal region of TcpC was required for efficient conjugation, oligomerization and protein-protein interactions. We conclude that by forming oligomeric complexes, TcpC contributes to the stability and integrity of the conjugation apparatus, facilitating efficient pCW3 transfer.