The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level. The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes. After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs. The groES cleavage product appeared to be unstable and subjected to degradation, while the groEL cleavage product appeared to be stable and became the major mRNA representing the groESL operon after long periods of growth at a high temperature. The polycistronic mRNA containing the groES and groEL genes was the major mRNA representing the groESL operon at a low temperature, and it reappeared when the cells were returned to the lower growth temperature after heat shock induction. These findings indicate that the cleavage event is part of the heat shock regulation of the groESL operon in A. tumefaciens.