The T‐DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. Upon initiation of the T‐DNA transfer process, site‐specific nicks occur at the 25‐bp border sequences. This cleavage leads to the generation of a free, linear ssT‐DNA molecule which is bound by sequence non‐specific VirE proteins. Here we present evidence for the involvement of other acetosyringone‐induced proteins in the formation of a covalent complex between the T‐strand and protein, designated the T‐complex. Alkaline gel‐electrophoretic analysis showed that proteins specifically bind to the 5′ termini of nicked T‐DNA molecules. The T‐complex can be formed in Escherichia coli when the VirD1 and VirD2 proteins are expressed.