Removal of extracellular Cl- has been shown to suppress light-evoked voltage responses of ON bipolar and horizontal cells, but not photoreceptors or OFF bipolar cells, in the amphibian retina. A substantial amount of experimental evidence has demonstrated that the photoreceptor transmitter, L-glutamate, activates cation, not Cl-, channels in these cells. The mechanism for Cl-free effects was therefore reexamined in a superfused retinal slice preparation from the mudpuppy (Necturus maculosus) using whole-cell voltage and current clamp techniques. In a Cl-free medium, light-evoked currents were maintained in rod and cone photoreceptors but suppressed in horizontal, ON bipolar, and OFF bipolar cells. Changes in input resistance and dark current in bipolar and horizontal cells were consistent with the hypothesis that removal of Cl- suppresses tonic glutamate release from photoreceptors. The persistence of light-evoked voltage responses in OFF bipolar cells, despite the suppression of light-evoked currents, is due to a compensatory increase in input resistance. Focal application of hyperosmotic sucrose to photoreceptor terminals produced currents in bipolar and horizontal cells arising from two sources: (a) evoked glutamate release and (b) direct actions of the hyperosmotic solution on postsynaptic neurons. The inward currents resulting from osmotically evoked release of glutamate in OFF bipolar and horizontal cells were suppressed in a Cl-free medium. For ON bipolar cells, both the direct and evoked components of the hyperosmotic response resulted in outward currents and were thus difficult to separate. However, in some cells, removal of extracellular Cl- suppressed the outward current consistent with a suppression of presynaptic glutamate release. The results of this study suggest that removal of extracellular Cl- suppresses glutamate release from photoreceptor terminals. Thus, it is possible that control of [Cl-] in and around photoreceptors may regulate glutamate release from these cells.