Phototropins (phot1 and phot2) are blue light (BL) receptors that mediate responses including phototropism, chloroplast movement and stomatal opening, and increased cytosolic Ca(2+). BL absorbed by phototropins activates plasma membrane H(+)-ATPase in guard cells, resulting in membrane hyperpolarization, and drives K(+) uptake and stomatal opening. However, it is unclear whether the phototropin-mediated Ca(2+) increase activates the H(+)-ATPase. Here, we determined cytosolic Ca(2+) concentrations in guard cell protoplasts (GCPs) from Arabidopsis transformed with aequorin. Cytosolic Ca(2+) increased rapidly in response to BL in GCPs from both the wild type and phot1 phot2 double mutants, but was mostly suppressed by an inhibitor of photosynthetic electron flow (DCMU). With depleted external K(+), we observed another slower Ca(2+) increase, which was phototropin- dependent. Fusicoccin, a H(+)-ATPase activator, mimicked the effect of BL. The slow Ca(2+) increase thus appears to result from membrane hyperpolarization. The slow Ca(2+) increase was suppressed by external K(+) and was restored by blockers of inward-rectifying K(+) channels, CsCl and tetraethylammonium, suggesting the preferential uptake of K(+) over Ca(2+). Such efficient K(+) uptake in response to BL was not found in mesophyll cells. Both the fast and the slow Ca(2+) increases were inhibited by Ca(2+) channel blockers (CoCl(2) and LaCl(3)) and a chelating agent (EGTA). These results indicate that the phototropin-mediated Ca(2+) increase was not observed prior to H(+)-ATPase activation in guard cells and that Ca(2+) entered guard cells via Ca(2+) channels through photosynthesis and phototropin-mediated membrane hyperpolarization.