Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn(2+) homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn(2+) physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn(2+) concentrations with high spatial and temporal fidelity. To address this critical need, we have synthesized, characterized, and applied an intracellular fluorescent probe for the ratiometric imaging of Zn(2+) based on a tautomeric seminaphthofluorescein platform. Zin-naphthopyr 1 (ZNP1) affords single-excitation, dual-emission ratiometric detection of intracellular Zn(2+) through Zn(2+)-controlled switching between fluorescein and naphthofluorescein tautomeric forms. The probe features visible excitation and emission profiles, excellent selectivity responses for Zn(2+) over competing Ca(2+) and Mg(2+) ions at intracellular concentrations, a dissociation constant (K(d)) for Zn(2+) of <1 nM, and an 18-fold increase in fluorescence emission intensity ratio (lambda(624)/lambda(528)) upon zinc binding. We demonstrate the value of the ZNP1 platform for biological applications by imaging changes in intracellular [Zn(2+)] in living mammalian cells. Included is the ratiometric detection of endogenous pools of intracellular Zn(2+) after NO-induced release of Zn(2+) from cellular metalloproteins. We anticipate that ZNP1 and related probes should find utility for interrogating the biology of Zn(2+).