The VirD2 protein of Agrobacterium tumefaciens is essential for processing and transport of the T-DNA. It has at least three functional domains: a relaxase domain at the N terminus, a bipartite nuclear localization signal (NLS), and a sequence called omega at the C terminus. We confirm here that deletions of the C-terminal part of VirD2 led to lack of transfer of T-DNA but, for the first time, we report that virulence is restored when these truncations are supplemented at the C terminus by a short translocation signal from the VirF protein. The lack of virulence of C-terminal deletions suggests that the C-terminal part contains all or part of the translocation signal of VirD2. Using a novel series of mutant VirD2 proteins, the C-terminal half of VirD2 was further investigated. We demonstrate that the C-terminal 40 amino acids of VirD2, which include the NLS and omega, contain all or part of the translocation domain necessary for transport of VirD2 into plant cells, while another element is present in the middle of the protein. The finding that a type IV secretion system transport signal at the C terminus of VirD2 is necessary for virulence provides evidence for the role of VirD2 as a pilot protein driving translocation of the T-strand.