Cell-to-cell communication evolved as a fundamental process to coordinate the physiological behavior in multicellular organisms. How cell behavior is regulated by the diverse signalling pathways, what mechanisms the cells use to distinguish the input from different receptors activating the same signals, are key questions in modern biomedical research that need to be addressed for a better understanding of physiology and physiopathology. To gain insights into the complexity of the spatio-temporal patterns of the signals that govern cell communication, we need tools that permit the visualization and quantitative measurement of such signals by fluorescent microscopic imaging in living cells. The spatial complexity of the cell and the spatiotemporal heterogeneity of signals require not only the quantitative determination of such signals in the cytoplasm of living cells, but also within subcellular compartments and organelles. Here I will discuss the approaches that have been developed in my laboratory to study second messenger heterogeneity, in particular Ca²⁺ and cyclic nucleotides, and the novel information obtained by the use of this methodologies in a few examples relevant to human pathology. Finally, I will concentrate on the mechanisms regulating second messenger homeostasis in mitochondria, an organelle that in the last few years has been at the centre of biomedical research for its participation not only in cell energy production, but also for its role as master regulator of programmed cell death.