The effect of free Ca2+ ion concentration on the integrity of the barrier function of the corneal endothelium was studied using the endothelial permeability to carboxyfluorescein (Pac) as a quantitative index according to the method of Araie. Paired rabbit corneas were isolated and mounted in a chamber. To serve as a control, one eye of each pair was perfused with a glucose-glutathione-bicarbonate solution at a Ca2+ concentration of 1.1 mEq, the other with a solution at various Ca2+ concentrations ranging from 0.23-1.1 mEq. The Pac ratio of a solution with a Ca2+ concentration of 0.38 mEq or higher to the control solution was close to unity, and the Pac ratio of a solution with a Ca2+ concentration of 0.33 mEq or lower to the control solution was significantly greater than unity. In a separate experiment, it was found that only slight swelling was seen when a solution with a Ca2+ concentration of 0.23 mEq or higher was used; significant swelling was seen with a solution of a Ca2+ concentration of 0.17 mEq or lower. The lowest free Ca2+ concentration needed for maintaining the barrier function of the corneal endothelium, 0.38 mEq, was found to be higher than that needed for maintaining the corneal thickness, 0.23 mEq.