Two new fluorescent sensors for Zn2+ that utilize fluorescein as a reporting group, Zinpyr-1 and Zinpyr-2, have been synthesized and characterized. Zinpyr-1 is prepared in one step via a Mannich reaction, and Zinpyr-2 is obtained in a multistep synthesis that utilizes 4 ' ,5 ' -fluorescein dicarboxaldehyde as a key intermediate. Both Zinpyr sensors have excitation and emission wavelengths in the visible range (similar to 500 nm), dissociation constants (K-d1) for Zn2+ of <1 nM, quantum yields approaching unity (Phi = similar to0.9), and cell permeability, making them well-suited for intracellular applications. A 3- to 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn2+ cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The X-ray crystal structure of a 2:1 Zn2+:Zinpyr-1 complex has also been solved, and is the first structurally characterized example of a complex of fluorescein substituted with metal binding ligands.