1. Blockage and permeation of divalent cations through channels activated by guanosine 3',5'-cyclic monophosphate (cyclic GMP) were studied in membrane patches excised from retinal rods of the tiger salamander Ambystoma tigrinum by rapidly changing the ionic medium bathing the intracellular side of the excised membrane. 2. The Na+ current, observed when 110 mM-NaCl was present on both sides of the membrane patch, was reduced by the addition of 1 mM of the chloride salts of Ca2+, Mg2+, Sr2+, Ba2+ or Mn2+ to the bathing medium. The sequence of blocking potency at +60 mV was Mg2+ greater than Mn2+ approximately Ba2+ greater than Ca2+ greater than Sr2+, while at -60 mV it was Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ approximately Mg2+. For all divalent cations the blocking effect depended, in a complex way, on the membrane potential. 3. The blocking effect of Ca2+ and Mg2+ increased when the concentration of cyclic GMP was reduced from 100 to 5 microM. At -60 mV 1 mM-Ca2+ blocked about 34% of the Na+ current in the presence of 100 microM-cyclic GMP, while in the presence of 5 microM-cyclic GMP, 1 mM-Ca2+ blocked about 56% of the Na+ current. 4. When, in the presence of 100 microM-cyclic GMP, 110 mM-NaCl at the intracellular side was replaced by equiosmolar amounts of chloride salts of divalent cations (73.3 mM) a small outward current carried by divalent cations could be observed at large positive membrane potentials. At +60 mV the ratio between the current carried by Na+, Sr2+, Ca2+, Ba2+, Mg2+ and Mn2+ was 83.3:1.4:1:0.58:0.33:0.25. 5. In agreement with previous observations the dependence of the Na+ current on the concentration of cyclic GMP shows a clear co-operativity among cyclic GMP molecules.4+ cyclic GMP-gated channel in excised patches is similar to but not identical to the selectivity sequence of divalent cations through the channel in intact rods.