A set of Agrobacterium tumefaciens operons required for pathogenesis is coordinately induced during plant infection by the VirA and VirG proteins. The intracellular concentration of VirG increases in response to acidic media, and this response was proposed to be regulated at the level of transcription at a promoter (P2) that resembles the Escherichia coli heat shock promoters. To test this hypothesis, we first constructed a virG-lacZ transcriptional fusion. A strain containing this fusion had higher levels of beta-galactosidase activity in acidic media than in media at neutral pH. Second, primer extension analysis of virG indicated that acidic media stimulated the transcription of this promoter. To determine whether P2 is a member of a heat shock-like regulon in A. tumefaciens, five agents that induce E. coli heat shock genes were tested for their abilities to induce a P2-lacZ fusion in A. tumefaciens. P2 was most strongly induced by low pH, was moderately stimulated by CdCl2 or mitomycin C, and was slightly induced by P2 as measured by beta-galactosidase activity and primer extension analysis. Induction by these treatments did not require any Ti plasmid-encoded function or the chromosomally encoded RecA protein. We also pulse-labeled cellular proteins after a shift to low pH and detected several proteins whose synthesis was induced by these conditions. We conclude that P2 is primarily induced by acid pH and secondarily by certain other stimuli, each of which is stressful to cell growth. This stress induction is at least partly independent of the heat shock and SOS responses.