Glutamate produces a hyperpolarizing synaptic potential in On bipolar cells by binding to the metabotropic glutamate receptor mGluR6, leading to closure of a cation channel. Here it is demonstrated that this cation channel is regulated by intracellular Ca(2+). Glutamate-evoked currents were recorded from On bipolar cells in light-adapted salamander retinal slices in the presence of 2 mm external Ca(2+). When glutamate was applied almost continuously, interrupted only briefly to measure the size of the response, the glutamate response remained robust. However, currents elicited by intermittent and brief applications of glutamate exhibited time-dependent run down. Run down of the glutamate response was also voltage dependent, because it was accelerated by membrane hyperpolarization. Run down was triggered, at least in part, by a rise in intracellular Ca(2+); measured as a function of time or voltage, it was attenuated by intracellular buffering of Ca(2+) with BAPTA or by omitting Ca(2+) from the bathing solution. Current-voltage measurements demonstrated that Ca(2+) induced run down of the glutamate response by downregulating cation channel function, rather than by preventing closure of the channel by glutamate and mGluR6. A major source of the Ca(2+) that mediated this inhibition is the cation channel itself, which was found to be permeable to Ca(2+), accounting for the use dependence of the run down. These results suggest that Ca(2+) influx through the cation channel during background illumination could provide a signal to close the cation channel and repolarize the membrane toward its dark potential, an adaptive mechanism for coping with changes in ambient light.