Electrophysiological studies of the isolated retina involve perfusing the tissue with a physiological Ringer's. Organic pH buffers such as HEPES have become increasingly popular in recent years because for many purposes they offer a convenient and reliable alternative to the more traditional bicarbonate/CO2. In this paper, however, we report that important functional properties of rods, bipolar cells, and horizontal cells in the salamander, Ambystoma tigrinum, are sensitive to the choice of buffer and, in the case of horizontal cells, that sensitivity is acute. In bicarbonate/CO2 Ringer's, the dark potential of the horizontal cell was typically near −50 mV and saturating light caused it to hyperpolarize to about −75 mV. On switching to HEPES-buffered Ringer's at the same pH, horizontal cells depolarized in darkness to about −20 mV, close to the chloride equilibrium potential, and the kinetics of their light responses changed. The cone-driven components of light responses increased in size relative to rod-driven components. Saturating lights still hyperpolarized the cells to −75 mV, however. Horizontal cells, being coupled via gap junctions, form a syncytium and syncytial length constants, measured in bicarbonate/CO2 Ringer's, were generally in the range 150–225 μm. On switching to HEPES-buffered Ringer's, length constants increased substantially to 250–330 μm. All these changes were reversible. We discuss our findings within the context of the cell's ability to regulate its internal pH.