A 68-kDa heat-stress protein (HSP68) has been purified from cell-suspension cultures of tomato (Lycopersicon peruvianum L.). Antibodies raised against HSP68 cross-react with the Escherichia coli heat-stress protein DnaK. HSP68 was found to be a hydrophilic, ATP-binding protein. Immunological analysis of subcellular fractions and immunogold-labelling of ultrathin sections showed consistently that HSP68 is localized in the mitochondrial matrix. In-vitro translation experiments indicated that HSP68 is synthesized as a precursor protein. Immunoscreening of cDNA libraries from tomato and potato (Solanum tuberosum L.) led to the isolation of corresponding cDNA clones. The deduced amino-acid sequences show strong relationships to the DnaK-like proteins from bacteria and organelles of eukaryotic cells. The protein HSP68 is constitutively expressed, but its synthesis is increased during heat stress in all cells of higher plants investigated so far.