We have examined the heat shock response of Rhizobium leguminosarum. After normal growth at 28 degrees C, a 10 min heat shock at 37 degrees C induced the synthesis of proteins with approximate M(r) values of 90,000, 70,000, 60,000, 58,000, 19,000, 17,000 and 13,000. A monoclonal antibody raised against the E. coli Cpn60 cross-reacted with proteins of M(r) 60,000 and 58,000 in R. leguminosarum, suggesting that both were Cpn60 homologues. Hybridization of an E. coli cpn60 probe to total DNA from Rhizobium leguminosarum also showed evidence for at least two cpn60 homologues. One of these was cloned and completely sequenced, and showed close homology to cpn60 sequences from other prokaryotes. The expression of this gene in E. coli failed to complement a cpn60 mutation, either for growth at high temperature or for growth of bacteriophage lambda. Hybridization of total R. leguminosarum DNA with a probe from this gene revealed the presence of a third putative cpn60 gene. Two further hybridizing clones were analysed and found to consist of two additional cpn60 sequences plus upstream regions containing putative cpn10 genes.