The 8-azapurines, and their 7-deaza and 9-deaza congeners, represent a unique class of isosteric (isomorphic) analogues of the natural purines, frequently capable of substituting for the latter in many biochemical processes. Particularly interesting is their propensity to exhibit pH-dependent room-temperature fluorescence in aqueous medium, and in non-polar media. We herein review the physico-chemical properties of this class of compounds, with particular emphasis on the fluorescence emission properties of their neutral and/or ionic species, which has led to their widespread use as fluorescent probes in enzymology, including enzymes involved in purine metabolism, agonists/antagonists of adenosine receptors, mechanisms of catalytic RNAs, RNA editing, etc. They are also exceptionally useful fluorescent probes for analytical and clinical applications in crude cell homogenates.