The light-sensitive current in photoreceptors is conducted by a single class of ion channels gated by the binding of multiple molecules of cytoplasmic cGMP. Both Na and Ca ions enter the outer segment through this channel and Ca behaves as a blocking ion, greatly reducing the influx of Na. Because intracellular Ca functions as the cytosolic messenger for light adaptation, and this channel is the major entry point for Ca into the outer segment, we seek a better understanding of the selectivity properties of the channel and how they affect intracellular Ca levels. In these studies, we added divalent cations to the cytoplasmic face of an excised patch at constant, symmetrical [Na]. Our results suggest a novel high-affinity divalent binding site at the internal face of the channel. At constant low levels of cGMP, the addition of 10-100 nM cytoplasmic Ca or Mg attenuated the current 5- to 10-fold. There is also a low-affinity site, midway through the transmembrane field; saturation of this site reduces the divalent-free current approximately 100-fold. The presence of a high-affinity cytoplasmic site raises the question of whether Ca regulates the photoreceptor current through a direct interaction with the channel perhaps altering the channel selectivity or kinetics.