In this study, Zn²⁺ levels in rat cortical neurons were measured by successfully combining radioactive tracer experiments with spectrofluorometry and fluorescence microscopy. Cortical neurons showed a time dependent and saturable transport of ⁶⁵Zn²⁺ with an apparent affinity of 15-20 µM. ⁶⁵Zn²⁺ transport was pH dependent and was decreased by extracellular acidification and increased by intracellular acidification. Compartmentalization of newly transported Zn²⁺ was assessed using the Zn²⁺ selective fluorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn²⁺. Depletion of intracellular Zn²⁺ using the membrane permeable chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), resulted in the complete loss of punctate zinquin labeling. After Zn²⁺ depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 µM Zn²⁺, pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 µM Zn²⁺ pH 6.0. These data were confirmed in parallel ⁶⁵Zn²⁺ transport experiments.