Subcellular fractionation and immunofluorescence microscopy were used to identify the specific sites of intracellular residence of mortalin, also called a mitochondrial homologue of the hsp70 family, in immortal human cell Lines previously assigned to four distinct complementation groups (A-D) for indefinite cell division. In addition to the mitochondria it was seen in the endoplasmic reticulum (ER) fractions of all the cell Lines analyzed. Interestingly, three of the group A cells lines (EJ, GM639, and HT1080), in addition to the mitochondria and ER, exhibited cytosolically (extra-organelle) localized pool of mortalin. These findings demonstrate that mortalin is not present exclusively in mitochondria Its residence in different organelles may be the basis of differential distribution observed previously in different human cell lines.