The H+ hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H+ release from horizontal cells. To test this hypothesis, self-referencing H+-selective microelectrodes were used to measure extracellular H+ fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H+ adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H+ that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H+. A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca2+ ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H+. These findings argue strongly against the hypothesis that H+ release from horizontal cells mediates lateral inhibition in the outer retina.