- Mitochondria are emerging as a major hub for cellular Ca(2+)-signaling, though their contribution to Ca(2+)-driven growth- and survival-promoting events in cancer is poorly understood. Here employing flow cytometry to monitor mitochondrial and cytosolic Ca(2+), we assessed trans-mitochondrial Ca(2+)-transport and store-operated Ca(2+)-influx (store-operated channels (SOC)) in malignant vs. non-malignant B16BL6 melanoma clones. Remarkably, mitochondrial Ca(2+)-fluxes measured in whole cells or in isolated mitochondria were accelerated in the malignant clones compared to their non-malignant counterpart clones. This coincided with enhanced SOC-mediated Ca(2+)-influx and high levels of constitutively active protein kinase B/Akt (PKB). Interruption of trans-mitochondrial Ca(2+)-transport in the malignant cells with an antagonist of the mitochondrial Na(+)/Ca(2+) exchanger, CGP-37157, abolsihed SOC-mediated Ca(2+)-influx, inactivated PKB, retarded cell growth and increased vulnerability to apoptosis. Similarly, direct SOC blockade by silencing Stim1 inhibited PKB, indicating that the crosstalk between SOC and mitochondria is essential to preserve PKB in constitutively active state. Finally, the retraction of mitochondria from sub-plasmalemmal micro-domains triggered by Fis1 over-expression inhibited SOC-coupled trans-mitochondrial Ca(2+)-flux, Ca(2+)-entry via SOC and PKB activity. Taken together, our data show that in the malignant melanoma cells, the functional and spatial relationship of up-regulated mitochondrial Ca(2+)-transport to the SOC sustains the robust Ca(2+)-responses and down-stream signaling critical for apoptosis-resistance and proliferation.