During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.