To investigate modulation of the activation of cGMP-gated ion channels in cone photoreceptors, we measured currents in membrane patches detached from the outer segments of single cones isolated from striped bass retina. The sensitivity of these channels to activation by cGMP depends on the history of exposure to divalent cations of the membrane's cytoplasmic surface. In patches maintained in 20 microM Ca++ and 100 microM Mg++ after excision, the current amplitude dependence on cGMP is well described by a Hill equation with average values of K1/2, the concentration necessary to activate half the maximal current, of 86 microM and a cooperativity index, n, of 2.57. Exposing the patch to a solution free of divalent cations irreversibly increases the cGMP sensitivity; the average value of K1/2 shifts to 58.8 microM and n shifts to 1.8. Changes in cGMP sensitivity do not affect other functional parameters of the ion channels, such as the interaction and permeation of mono- and divalent cations. Modulation of cGMP activation depends on the action of an endogenous factor that progressively dissociates from the channel as Ca++ concentration is lowered below 1 microM. The activity of the endogenous modulator is not well mimicked by exogenously added calmodulin, although this protein competes with the endogenous modulator for a common binding site. Thus, the modulation of cGMP affinity in cones depends on the activity of an unidentified molecule that may not be calmodulin.