Mortalin, a novel member of mouse heat shock protein 70 (hsp70) family, is seen to distinguish the cellular mortal and immortal phenotypes by virtue of its cytosolic and perinuclear distribution, respectively. We report here that the cytosolic and perinuclear forms of mortalin from CD1-ICR mouse embryonic fibroblasts and NIH 3T3 cells, respectively, differ by two amino acids, can be distinguished on two-dimensional SDS-polyacrylamide gel. The perinuclear mortalins from RS-4 and Balb/c 3T3 cells harbor the same two amino acids as that of NIH 3T3 cells. However, these when analyzed with C-MEF mortalin did not exhibit the mobility shift equivalent to C-MEF and NIH 3T3 mortalins. The data indicate that the perinuclear mortalin from different immortal fibroblasts are not identical and implicate the possibility of additional structural changes in mortalin during immortalization. Such differences may also contribute to the differential in vitro growth characteristics of these immortal cells.