The ability of the divalent cations calcium, magnesium, and barium to permeate through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions and by measuring their ability to block current carried by sodium when presented on the cytoplasmic or extracellular side of the channel. Current carried by divalent cations in the absence of monovalent cations showed the typical rectification pattern observed from these channels under physiological conditions (an exponential increase in current at both positive and negative voltages). With calcium as the reference ion, the relative permeabilities were Ca > Ba > Mg, and the chord conductance ratios at +50 mV were in the order of Ca approximately Mg > Ba. With external sodium as the reference ion, the relative permeabilities were Ca > Mg > Ba > Na with chord conductance ratios at +30 mV in the order of Na >> Ca = Mg > Ba. The ability of divalent cations presented on the intracellular side to block the sodium current was in the order Ca > Mg > Ba at +30 mV and Ca > Ba > Mg at -30 mV. Block by external divalent cations was also investigated. The current-voltage relations showed block by internal divalent cations reveal no anomalous mole fraction behavior, suggesting little ion-ion interaction within the pore. An Eyring rate theory model with two barriers and a single binding site is sufficient to explain both these observations and those for monovalent cations, predicting a single-channel conductance under physiological conditions of 2 pS and an inward current at -30 mV carried by 82% Na, 5% Mg, and 13% Ca.