We reported a rapid, light-stimulated release of calcium from isolated rod outer segments that is apparent only when both the disk membrane and the plasma membrane are made permeable to calcium by adding the ionophore A23187 [Kaupp, U. B., Schnetkamp, P. P. M., & Junge, W. (1979) Biochim. Biophys. Acta 552, 390-403]. In this paper, we have investigated the light-sensitive diskal binding sites and the calcium release mechanism in their dependence on the pH and the presence of mono- and divalent cations, including calcium itself. We have observed now that several different rod outer segment preparations (i.e., rod outer segments with an intact plasma membrane, broken cells, and sonicated material) possess a similar dependence of their calcium release on the ionic conditions, however, only if manipulated in a way that gives access to the outer conditions of sites within disks (namely, ionophore added in the case of intact rod outer segments). Monovalent cations, at concentrations between 20 and 40 mM, suppress light-induced calcium release. Divalent and trivalent cations are more efficient inhibitors by 1-2 and 2-3 orders of magnitude, respectively. These results suggest that calcium release is controlled by an interfacial potential. The optimum pH for calcium release is pH 6.3, and virtually no release occurs beyond pH 4.5 and 9. The drop for acidic pH is attributed to the pH dependence of calcium binding to disk membranes, and the drop for alkaline pH is attributed to the pH dependence of the metarhodopsin I/metarhodopsin II transition and the light-stimulated proton uptake. In general, calcium release parallels calcium binding as a function of pH and calcium concentrations, although the release saturates at lower calcium concentrations ((KDapp = 5 microM) than would be expected from the amount of calcium bound (KD = 30-60 microM). The maximum stoichiometry is approximately 1 mol of calcium release per mol of rhodopsin bleached. Concomitant measurements of the light-stimulated uptake of protons by the disk membrane revealed a maximal stoichiometry of 2.8 mol of protons taken up per mol of rhodopsin bleached. We present an integrated description of light-stimulated calcium release, proton uptake, and changes of the interfacial potential at the disk membrane.