We have characterized the heat shock response of Agrobacterium tumefaciens and compared it with the well-characterized Escherichia coli heat shock response. Four major heat shock proteins with apparent molecular masses of 98, 75, 65, and 20 kDa were identified by pulse-labelling cultures after temperature upshift. The three largest proteins comigrated with proteins that were antigenically related to the E. coli heat shock proteins sigma 70, DnaK, and GroEL, respectively. The heat shock proteins were also strongly induced by ethanol and cadmium chloride and were mildly induced by mitomycin C. To determine whether the A. tumefaciens heat shock regulatory system was similar to that of E. coli, we introduced the E. coli dnaK gene into A. tumefaciens. The E. coli DnK protein was expressed in A. tumefaciens, and its synthesis was induced after heat shock. Primer extension analysis of the E. coli dnaK gene in A. tumefaciens indicated that transcription initiated from one or possibly both of the E. coli heat shock promoters. We conclude that A. tumefaciens has a heat shock response similar to that of E. coli, in that (i) similar proteins are induced by heat shock, (ii) synthesis of these proteins is induced in response to similar stimuli, and (iii) A. tumefaciens can recognize an E. coli heat shock promoter, suggesting that A. tumefaciens has a sigma factor similar to sigma 32.