The complete set of groES and groEL gene homologues from Bacillus subtilis Marburg 168 was identified, cloned, and characterized. The nucleotide sequence indicated the presence of two open reading frames corresponding to the groES and groEL genes. The presumptive GroES and GroEL proteins were calculated to be polypeptides of 10,175 and 57,175 Da, respectively, and showed extensive sequence similarities with the known GroES and GroEL proteins of Escherichia coli and Mycobacterium tuberculosis. A heat-inducible transcript initiated upstream of the groES coding region was identified by primer-extension analysis of in vivo transcripts, indicating that the two genes consist of an operon. At least six heat-shock inducible proteins were identified in the cell extract of heat treated B. subtilis. Two proteins of 10 and 60 kDa overproduced in B. subtilis cells carrying a multi-copy groES and groEL plasmid were demonstrated to correspond to two out of the six heat-shock inducible proteins. The groES and groEL genes of B. subtilis were physically mapped on the 60 degrees region of a 360 degrees map and genetically mapped at the position of 40% linkage with the purB locus using PBS1 transduction of the groEL genes tagged with a chloramphenicol resistance (chlr) marker.