ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE's interactions with its distinct protein partners.