Background light reduces the gain of phototransduction in retinal rods so that the ability to register changes in light intensity is not prevented by saturation of the cell's response. The gain is reduced by a light-induced fall in the intracellular calcium concentration which results from blockage of Ca2+ entry through the channels closed by light and continued Ca2+ extrusion by the Na:Ca,K exchanger. Calcium seems to exert several coordinated effects on the cyclic GMP cascade: a fall in [Ca2+] stimulates cGMP synthesis, increases the affinity of the cGMP-gated channel for cGMP and accelerates rhodopsin deactivation by phosphorylation. We now report that lowering intracellular [Ca2+] reduces the catalytic rhodopsin activity produced by light. The effect is operationally equivalent to a fourfold reduction in the number of rhodopsin molecules available for activation. The reduction in gain is cooperative and half-maximal at about 35 nM Ca2+, suggesting that it is mediated by a specific Ca(2+)-binding protein. Reduced rhodopsin activity in low Ca2+ should contribute to adaptation in background light.