Calcium acts as a second messenger in vertebrate rods, regulating the recovery phase of the light response and modulating sensitivity during light-adaptation. Since light not only decreases the outer segment calcium concentration (Ca²⁺_i) by closing cyclic nucleotide-gated channels but can also increase Ca²⁺_i by releasing it from buffer sites or intracellular stores, we examined in detail the effect of light and circulating current on Ca²⁺_i by making simultaneous measurements of suction pipette current and Ca²⁺_i from isolated rods of the salamander Ambystoma tigrinum after incorporation of the fluorescent dye fluo-5F. When the release of Ca²⁺ is measured in 0Ca²⁺/0Na⁺ solution, minimising fluxes of Ca²⁺ across the plasma membrane, it is substantial only for light bright enough to bleach a significant fraction of the photopigment and is restricted to the part of the outer segment in which the bleach occurred. It is unlikely, therefore, to make a large contribution to Ca²⁺_i for most of the physiological operating range of the rod. Nevertheless, since release is half maximal for a bleach of less than 10%, it cannot be produced by a simple mechanism such as a change in the affinity of a binding site on rhodopsin itself but must instead require some more complex interaction. In Ringer solution, the Ca²⁺ in the light-releasable pool can be discharged merely by the decrease in Ca²⁺_i that occurs as the outer segment channels close. In steady background light or after exposure to saturating illumination, the fraction of Ca²⁺ in the pool decreases essentially in proportion to Ca²⁺_i as if Ca²⁺ were being removed from a buffer site within the cytoplasm. Furthermore, Ca²⁺_i itself changes in proportion to the circulating current, with little evidence for a contribution from Ca²⁺ release or other mechanisms of Ca²⁺ homeostasis. This indicates that flux of Ca²⁺ across the plasma membrane is the major determinant of outer segment Ca²⁺ concentration within the rod's normal operating light intensity range. Once Ca²⁺ has been discharged from the releasable pool, it is restored following dim illumination apparently as the simple result of the subsequent restoration of dark Ca²⁺_i and the rebinding of Ca²⁺ to its release site, but after brighter light perhaps also as a consequence of regeneration of the photopigment.