Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants. VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore. To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes. In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence microscopy, cells probed with anti-VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location of VirD4 was not dependent on T-DNA processing, the formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large cytoplasmic region contains a nucleotide-binding domain. To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer. A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer. Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide-binding domains are required for polar localization of VirD4. The periplasmic domain mutant VirD4Δ36-61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.