The columella cells of soybean roots grown under gravity and simulated microgravity induced by a clinostat were examined using potassium pyroantimonate (PA) and quantitative X-ray microanalysis of cryosections to determine the role of Ca in the regulation of the gravitropic response. Amyloplasts in the columella cells were localized exclusively at the bottom under gravity, but diffusely distributed in the cytoplasmic matrix under simulated microgravity, thus supporting the statolith theory. In the columella cells, PA precipitates containing Ca were diffusely distributed in the cytoplasmic matrix under gravity. Under simulated microgravity, however, they decreased in number and size in the cytoplasmic matrix, whereas increased only in number in the vacuole, indicating that Ca moved from the cytoplasmic matrix into the vacuole. The vacuole of columella cells contained mostly electron-dense granular structures localized along the inner surface of tonoplasts, which closely resembled the tannin vacuole reported in Mimosa pulvinar motor cells. Under simulated microgravity, their configuration changed dramatically from a granular shape to a flat plate. The quantitative X-ray microanalysis of cryosections showed that the vacuolar electron-dense structures contained a large amount of Ca. Under simulated microgravity, the concentration of Ca increased conspicuously in these vacuolar electron-dense structures, concomitantly with a marked decrease of K in the vacuoles and an increase of K in the cell walls. These results suggest that the release of Ca²⁺ from, and uptake by, the vacuolar electron-dense structures is closely related to the signal transmission in the gravitropic response and that Ca movement occurs opposite to that of K.