The roles of 1) inactivation of Na-Ca+K exchange and 2) Ca2+ release from discs in regulation of cytosolic free Ca2+ were examined in intact rod outer segments (ROS) purified from bovine retinas. Measurements of cytosolic free Ca2+ (with fluo-3) were combined with Ca2+ flux measurements (45Ca) in ROS that contained about 600 microM total Ca2+. Na(+)-induced Ca2+ extrusion was measured in a Ca(2+)-free medium and did not lower cytosolic free Ca2+ to below 1 nM as expected from a coupling stoichiometry of 4Na+:(1Ca(2+) + 1K+). Instead, cytosolic free Ca2+ was rapidly (20 s) lowered from about 1300 nM to 100-150 nM, while at the same time about 35% of total ROS Ca2+ was removed. During the next 40 min cytosolic free Ca2+ remained virtually steady, but total ROS Ca2+ was reduced by a further 50% at a 100-fold lower rate than that observed for the initial fast phase. The steady cytosolic Ca2+ concentration resulted from Ca2+ release from discs and subsequent removal across the plasma membrane by Na-Ca+K exchange operating at a greatly reduced rate. Addition of the alkali cation channel ionophore gramicidin led to a persistent increase in cytosolic free Ca2+ concentration to about 400 nM, presumably caused by an increase in intracellular Na+. It is suggested that cytosolic free Ca2+ is not determined by the Na+:Ca2+ coupling ratio of the exchanger, but rather by a sensor on its cytoplasmic domain that controls inactivation of the Ca2+ extrusion mode and is sensitive to intracellular Ca2+, Na+, and K+.