Induction of Ti plasmid virulence (vir) genes during early stages of the genetic transformation of plant cells by Agrobacterium tumefaciens results in several molecular events that are involved in generating a transferable T-DNA copy. These events include site-specific nicking at the T-DNA borders and synthesis of free, unipolar, linear, single-stranded copies of the T-DNA (T-strands). Here E. coli was used as a heterologous cell to assay the requirements for T-strand synthesis. Cells of E. coli harbored two compatible plasmids, one containing coding sequences overlapping the virC and virD regions of the nopaline Ti plasmid, and a second plasmid containing a T-DNA region. The amount of vir proteins produced was varied by placing their expression under the control of either native Agrobacterium, tac, or T7 promoters. The data show that VirD1 and VirD2 proteins are absolutely essential for T-strand production in E. coli, and the relative amounts of these polypeptides produced correlate with the amounts of T-strand observed. When VirD1 and VirD2 products are limiting, the VirC1 protein increases T-strand production. The yield of T-strands also varies as a function of the plasmid vector used to clone the T-DNA region substrate; the same T-DNA cloned into pLAFR1 produces more T-strands than that cloned into the higher copy number plasmid pACYC184. In summary, VirD1 and VirD2 proteins are the minimal requirements for T-strand production; however, other factors such as VirC1, the relative concentration of VirD1, VirD2, and the T-DNA substrate, and possibly additional functions (e.g., those specified by pLAFR1) influence the efficiency of T-strand production. Additional results regarding the requirements for expression of VirD1 and VirD2 polypeptides are presented.