Intracellular Ca²+ is involved in a larger series of cell and physiological processes in health and disease including cell proliferation, cell migration and cell death. Intracellular calcium has been selected by nature as the most pleiotropic and extended second messenger. Calcium ions are not synthesized or destroyed like other messengers. Instead, they are transported up and down electrochemical gradients through a series of transport systems (calcium channels, pumps and co-transporters) located in plasma membrane and endomebranes of the endoplasmic reticulum, mitochondria, Golgi and nuclear envelop. The use of sophisticated approaches of Cell Physiology as Calcium Imaging and Patch Clamp electrophysiology allows to monitor, in real time, changes in the levels of cytosolic and subcellular calcium together with the activity of ion channels. The combination with Molecular Biology approaches including qRT-PCR, western blotting, immunofluorescence, sirRNA and next generation sequenting is enabling the discovery of many important roles of intracellular calcium and calcium transporters in health and disease. We use these approaches to investigate the role of calcium in cancer, aging and Alzheimer´s disease.