The loss of Mg(2+) upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 millimolar MgCl(2) with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 millimolar MgCl(2) if the chloroplasts were in a K(+) -gluconate medium. Measurements of stromal pH in the dark indicated that, in the K(+) -gluconate medium, the stromal pH is nearly the same as that of the medium, whereas in the sorbitol medium it is much more acidic as reported previously. These observations suggest that the free Mg(2+) concentration in the stroma in the dark is between 1 and 3 millimolar. Other experiments on the inihibition by A23187 of CO(2) fixation in the light and in a system capable of catalyzing CO(2) fixation in the dark, and on the Mg(2+) binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mg(2+) concentration changes occur in the stroma that are important in the light-dark regulation of CO(2) fixation.