The purines, adenosine triphosphate (ATP) and adenosine, are now recognized as extracellular signaling agents in both the brain and peripheral organs. The mechanisms of purinergic signaling can differ from those of conventional neurotransmission especially in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real time measurements of these purinergic agents have therefore been of great value in elucidating the functional roles of ATP and adenosine in several diverse contexts. This review introduces the concept of microelectrode biosensors for ATP and adenosine and explains how they operate, how they can best be used, and gives examples of new mechanistic insight into the mechanosensory control of respiration, synaptic physiology, and glucosensing in the hypothalamus.