Using the H(+)-sensitive fluorophore 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and microfluorimetry, we investigated how elevated intracellular free zinc ([Zn(2+)](i)) altered intracellular proton concentration (pH(i)) in dissociated cultures of rat forebrain neurons. Neurons exposed to extracellular zinc (3 microM) in the presence of the Zn(2+)-selective ionophore pyrithione (20 microM) underwent intracellular acidification that was not reversed upon washout of the stimulus. Application of a membrane-permeant Zn(2+) chelator, but not an impermeant chelator, partially restored pH(i). Removal of extracellular Ca(2+) greatly inhibited [Zn(2+)](i)-induced acidification, suggesting that acidification was a secondary consequence of Ca(2+) entry. Additional experiments suggested that Ca(2+) entered through the plasma membrane sodium/calcium exchanger (NCE), because a specific inhibitor of reverse mode NCE operation, KB-R7943 (1 microM), significantly inhibited Zn(2+)-induced acidification. In addition to the phenomenon of [Zn(2+)](i)-induced acidification, we found that elevated [Zn(2+)](i) inhibited neuronal recovery from low pH(i). Neurons exposed to a protonophore underwent robust acidification, and pH(i) recovery ensued upon protonophore washout. In contrast, neurons acidified by the protonophore in the presence of Zn(2+) (3 microM) and pyrithione (20 microM) showed no ability to recover from low pH(i). Application of a membrane-permeant Zn(2+) chelator partially restored pH(i) to pre-stimulus values. Experiments designed to elucidate mechanisms responsible for pH(i) regulation revealed that neurons relied primarily on bicarbonate exchange for proton export, suggesting that elevated [Zn(2+)](i) might impede pH(i) by inhibiting proton efflux via bicarbonate exchange. These results provide novel insights into the physiological effects of raising [Zn(2+)](i), and may help illuminate the mechanisms by which Zn(2+) injures neurons.