Mortalin/mtHSP70 and HSP60 are heat-shock proteins that reside in multiple subcellular compartments, mitochondria being the dominant compartment. We present here biochemical evidence for their in vivo and in vitro interactions. By the use of quantum dots (powerful tools used for simultaneous imaging of multiple proteins), we visualized minute differences in the subcellular niche of these two proteins in normal and cancer cells. Knockdown of either of these two by shRNA expression plasmids caused growth arrest of osteosarcoma cells. However, interestingly, whereas an overexpression of mortalin extended in vitro life span of normal fibroblasts (TIG-1), overexpression of HSP60 was neutral. We demonstrate the minute differences in subcellular distribution of mortalin and HSP60, their involvement in tumorigenesis, and functional distinction in pathways involved in senescence.