The positive regulatory protein VirG from the virulence region of the Ti plasmid of Agrobacterium tumefaciens was first demonstrated to possess DNA-binding capabilities using chromatographically purified protein and in vitro assays (Powell et al., 1989). This paper is an extension of that research and presents evidence on the in vivo DNA-binding properties of VirG using a transcription interference assay. VirG protein bound specifically to a 'vir box' response element and repressed transcription of a lacZ reporter gene, but increased transcription in the absence of a vir box. A biphasic response in specific DNA-binding was observed upon increasing virG expression, suggesting that specific binding was co-operatively affected by protein concentration. Certain TrpE'-'VirG hybrid proteins also bound the vir box, but required sequences distal to amino acid Arg-118 of the VirG polypeptide. These data further localize a DNA-binding domain within VirG, and support a modified model for the regulation of virulence genes in which transphosphorylation by the coregulator VirA functions to stabilize specific DNA-binding by low concentrations of VirG, resulting in gene activation. Otherwise, at high concentrations, VirG promotes expression of the virulence regulon without assistance from VirA as was shown previously (Rogowsky et al., 1987).