We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K1/2, also increases as Ca2+ concentration decreases. In electropermeabilized cones, K1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca2+, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca2+ and 15.3 ± 4.5 μM in its absence. The Ca2+-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca2+ and 7.2 ± 1.2 μM in its absence. The Ca2+-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca2+-dependent change in sensitivity in cones is half-maximal at KCa = 286 ± 66 nM Ca2+. In rods, by contrast, KCa is ∼50 nM Ca2+. The difference in magnitude and Ca2+ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.