Octopine and nopaline strains of Agrobacterium tumefaciens were found to differ in virulence on Nicotiana glauca. This difference is due to the absence of a functional virF locus, which is necessary for efficient tumorigenesis on N. glauca, from the nopaline Ti plasmids. Genetic studies and DNA sequence analysis of the virF locus revealed that virF embraces one open reading frame coding for a hydrophilic protein with a molecular mass of 22437 Da. Transcription of virF is directed from left to right, towards the T region, and is strongly induced by the phenolic compound acetosyringone. We established that virA and virG, two genes known to be essential for induction of the vir regulon, are necessary for acetosyringoneinduced virF expression, implying that virF is a member of this vir regulon. Agrobacterium virF mutants can be complemented for tumor induction by co-infection with avirulent Agrobacterium helper strains. We found that such helper strains must express not only the virF gene but also the vir operons virA, virB, virD and virG.