TnphoA mutagenesis of Agrobacterium tumefaciens identified new extracytoplasmic protein-encoding virulence loci. Mutations in these loci conferred increased sensitivity to detergents and several antibiotics. Clones carrying these loci were isolated from an A. tumefaciens cosmid library by complementation of the detergent sensitivities of the mutants. The locus on one complementing clone was delineated by Tn5 and TnphoA mutagenesis. DNA sequence analysis of the delineated region revealed that this locus is made up of two transcriptional units, chvG and chvI, which were predicted, on the basis of amino acid sequence homology, to encode the members of a two-component sensory transduction system. The membrane-spanning sensor, a histidine protein kinase, was designated ChvG, and the response regulator, presumably a transcriptional activator, was designated ChvI. Surprisingly, ChvG was also predicted to contain a Walker type A consensus nucleotide binding site, which is unusual for sensor histidine protein kinases. Site-specific insertion mutations in either chvG or chvI abolished tumor formation ability, as well as the ability to grow on complex media. Neither the genes which are regulated nor the inducing signal is known yet for this system.