We report the cloning, nucleotide sequence, and localization of mitochondrial hsp70, a member of the human hsp70 multi-gene family. The human mthsp75 gene was cloned by screening an expression library with monoclonal antibody 3A3 that recognizes three members of the human hsp70 family (hsp70, hsc70, and a 75-kDa protein with characteristics identical to that previously established for mitochondrial hsp70). The identity of the 75-kDa protein was confirmed by subcellular fraction of HeLa cells and the demonstration that the 3A3-reactive 75-kDa protein co-fractionates with mitochondrial localized proteins. The nucleotide sequence of the respective cDNA clone revealed an open reading frame of 679 amino acids with extensive sequence identity with members of the human hsp70 family. The derived amino-terminal pre-sequence shares features common to other mitochondrial targeting sequences. The identity of the cDNA was unequivocally established by introduction of an epitope-tag at the carboxyl terminus of the cloned gene, transfection and analysis by immunofluorescence. The tagged 75-kDa protein localizes to mitochondria, thus providing conclusive evidence that it corresponds to the human mitochondrial hsp70, referred to here as mthsp75.