Members of the 70-kD heat shock protein family have been found in all free-living organisms investigated and in major compartments of eukaryotic cells where they are essential to a wide range of functions, including protein folding and targeting. We have isolated a mitochondrial homolog (mtHSP70) from rat liver using ATP agarose affinity chromatography. Its identity was confirmed on the basis of immunological analysis and Ca(2+)-dependent autophosphorylation. Using protein sequence obtained from the amino termius and nine endo Lys-C peptide fragments, we have employed oligonucleotides to isolate a full-length cDNA clone. The open reading frame encodes a protein of 679 amino acids and calculated M(r) 73,913 daltons. The sequence has a high degree of identity with other members of the HSP70 family, including Escherichia coli DnaK (51%), Saccharomyces cerevisiae SSC1p (65%), the constitutive cytosolic HSP70 from rat, HSC70 (46%), and the rat endoplasmic reticulum isoform, BiP, (49%). The cDNA encodes a precursor protein with a 46-amino-acid signal peptide that is absent from the protein isolated from rat liver. The protein also shows a high degree of identity (98%) with a protein isolated from mouse and human tissues (PBP74, Domanico et al., 1993; mortalin, Wadhwa et al., 1993a; CSA, Michikawa et al., 1993a); however, the intracellular localization of these proteins is uncertain. We show that the precursor of mtHSP70 is efficiently imported into isolated mitochondria from rat liver and processed from 74 kD to the mature 69-kD protein.