A B S T R A C T We have investigated, with osmotic techniques, the light-regulated Na + channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na + permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na + channels are highly sensitive to membrane oxidation. Using the continuous flow technique, we investigated the H + and Ca ++ dependence of the Na + channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H + activity decreases P~a (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca ++ across membranes, the Na + channels are blocked when the external Ca ++ activity is increased from 10 -7 to 10 -s M. This high sensitivity to both H + and Ca ++ ions suggests that high field strength anionic sites may exist in or near the Na + channels and that the channels are blocked when these sites bind H + or Ca ++ ions.