The light-dependent pH changes in the suspending medium of guard cell protoplasts (GCP) from Vicia faba were studied. Upon illumination, the medium was initially slightly alkalinized and then acidified. The extent of alkalinization was lower in CO(2)-free air than in normal air. This initial alkalinization was inhibited by DCMU. Acidification in CO(2)-free air became observable in shorter duration of light exposure than that in normal air. The rate of acidification was higher in CO(2)-free air than in normal air. The CO(2) level of the medium decreased in the light, and increased in the dark. (14)CO(2) uptake was enhanced 2- to 3-fold by light, but not in the presence of DCMU. These results indicate that photosynthetic CO(2) fixation does take place in GCP and that the initial alkalinization is due to this photosynthetic CO(2) uptake. Diethylstilbestrol, a nonmitochondrial membrane-bound ATPase inhibitor, inhibited the acidification, suggesting that the acidification resulted from H(+) extrusion by GCP. The acidification in light was also prevented by KCN, and partly by DCMU. Possible mechanisms of alkalinization and acidification are discussed in relation to guard cell metabolism.