To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.