Phase separation of giant vesicles composed of neutral saturated lipid, negatively charged unsaturated lipid, and cholesterol, is observed at different calcium concentrations. Confocal microscopy provides the information where the phase separation becomes distinct as the calcium concentration is increased. The charged domains tend to bud toward the interior of the vesicle. This budding is assumed to be due to an increase in the osmotic pressure, in cooperation with the spontaneous curvature change in the outer leaflet of the bilayer caused by the adsorption of calcium ions and charge screening effect. We interpret the effect of small cations on the phase separation based on the theoretical model with the Poisson-Boltzmann equation. (C) 2010 Elsevier B.V. All rights reserved.