The metabolism of the chromophore of rhodopsin in the cytosol compartment of isolated intact cattle rod outer segments was used as an indicator for changes of the cytosolic Mg2+ and Ca2+ concentration upon changes of the external Mg2+ and Ca2+ concentration. The reduction of retinal to retinol upon photolysis of rhodopsin in situ in intact rod outer segments was critically dependent on the availability of cytosolic Mg2+. The latter is necessary as chelator of endogenous adenosine 5'-triphosphate (ATP). Lowering the cytosolic Ca2+ concentration beneath 10(-7) M resulted in an inhibition of the rate of retinol formation. This is presumably due to a light-activated process, which competes with retinol formation for the supply of high-energy phosphate from a common pool. These results led to the following conclusions. Changes of the external Mg2+ concentration are only followed by substantial changes of the cytosolic Mg2+ concentration when the ionophore A23187 is present. Changes of the external Ca2+ concentration are followed by parallel changes of the cytosolic Ca2+ concentration either when external Na+ is present or in the presence of A23187. Li+ and K+ could not substitute for Na+ in the former case, but K+ diminished the effectivity of Na+ at low Na+ concentrations and enhanced it at high Na+ concentrations. It is concluded that the control of cytosolic Ca2+ concentration in isolated intact rod outer segments is predominantly provided for by Na-Ca exchange, i.e., by coupled fluxes.