Calcium ions are ubiquitous and versatile signaling molecules, capable of decoding a variety of extracellular stimuli (hormones, neurotransmitters, growth factors, etc.) into markedly different intracellular actions, ranging from contraction to secretion, from proliferation to cell death. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca²⁺] rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. In this review, we discuss the structural and functional bases that generate the subcellular heterogeneity in cellular Ca²⁺ levels at rest and under stimulation. This complex choreography requires the concerted action of many different players; the central role is, of course, that of the calcium ion, with the main supporting characters being all the entities responsible for moving Ca²⁺ between different compartments, while the cellular architecture provides a determining framework within which all the players have their exits and their entrances. In particular, we concentrate on the molecular mechanisms that lead to the generation of cytoplasmic Ca²⁺ microdomains, focusing on their different subcellular location, mechanism of generation, and functional role.