Despite the key role of magnesium in many fundamental biological processes, knowledge about its intracellular regulation is still scarce, due to the lack of appropriate detection methods. Here, we report the spectroscopic and photochemical characterization of two diaza-18-crown-6 hydroxyquinoline derivatives (DCHQ) and we propose their application in total Mg2+ assessment and in confocal imaging as effective Mg2+ indicators. DCHQ derivatives 1 and 2 bind Mg2+ with much higher affinity than other available probes (K-d = 44 and 73 mu M, respectively) and show a strong fluorescence increase upon binding. Remarkably, fluorescence output is not significantly affected by other divalent cations, most importantly Ca2+, or by pH changes within the physiological range. Evidence is provided on the use of fluorometric data to derive total cellular Mg2+ content, which is consistent with atomic absorption data. Furthermore, we show that DCHQ compounds can be effectively employed to map intracellular ion distribution and movements in live cells by confocal microscopy. A clear staining pattern consistent with known affinities of Mg2+ for biological ligands is shown; moreover, changes in the fluorescence signal could be tracked following stimuli known to modify intracellular Mg2+ concentration. These findings suggest that DCHQ derivatives may serve as new tools for the study of Mg2+ regulation, allowing sensitive and straightforward detection of both static and dynamic signals.