ATP is a fundamentalmetabolic regulator present in virtually all cells and tissues, exerting wide-ranging biological effects through both intracellular and extracellular mechanisms. The physiological functions of extracellular ATP involve a sophisticated network of interrelated mechanisms, including the release of cellular ATP, the activation of purinergic receptors, the breakdown of ATP by ectoenzymes, and the deamination or cellular reuptake of adenosine and other nucleosides. In recent years, it has become increasingly evident that ATP release and intricate autocrine and paracrine purinergic signaling mechanisms are crucial for the regulation of a broad spectrum of immunomodulatory, neuromodulatory, prothrombotic, vasoactive, angiogenic, and many other cell functions. Here, we provide an overview of the primary ATP-release pathways and their roles in initiating purinergic signaling events at various physiological and pathological conditions. We also summarize recent advances in the detection of ATP levels in different cellular compartments and real-time monitoring techniques of its release into the extracellular space.