Novel fluorescent Mg2+ probes, 2'-carboxyfluorescein (2'-CF) and its derivatives, that are excitable by visible light, were designed, synthesized and characterized. After complexation with Mg2+, the absorption and emission maxima of these fluorescent probes were red-shifted and the fluorescence intensity was increased 11-fold at neutral pH. The apparent dissociation constant (Kd) of the Mg2+-2'-CF complex was 15.8 mM at neutral pH. The Kd of the Ca2+-2'-CF complex was about 10 times larger than that of the Mg2+ complex and the other derivatives showed similar characteristics. In contrast, all the commercially available fluorescent Mg2+ probes have a higher affinity for Ca2+ than Mg2+. 2'-CF fluoresced in alkaline solution (pH > 8) and the pK, value was 8.8. The pKa value of the Mg2+-2'-CF complex was 6.8, and the fluorescence intensity was increased in the neutral conditions. Thus, the addition of Mg2+ resulted in a lowering of the pKa, and also an increase of the fluorescence intensity.