In contrast to the E. coli chaperones DnaK, GroEL and GroES, cDNAs encoding mitochondrial homologues of DnaJ and GrpE from higher eukaryotes have yet to be reported. Based on peptide sequences, we have isolated a cDNA encoding a 217 residue nuclear encoded precursor of rat mitochondrial GrpE (mt-GrpE) including a typical mitochondrial presequence of 27 residues. Western blotting revealed that the 21 kDa GrpE homologue is present exclusively in the mitochondrial fraction where it comprises only approximately 0.03% of the total soluble protein, while Northern blotting showed that the mt-GrpE transcript is present in most if not all organs. By contrast to other mitochondrial chaperones, the levels of mt-GrpE and its transcript in cultured cells are only marginally increased in response to the proline analog L-azetidine 2-carboxylic acid but not by heat shock. Furthermore, members of the GrpE family exhibit a much lower degree of sequence identity than do the well studied members of the Hsp70, Hsp60 and Hsp10 families.