In the aleurone cells of the cereal grain, gibberellic acid (GA) induces the secretion of hydrolases that mobilize endosperm reserves to fuel early seedling growth. GA is known to trigger a range of cellular responses, including increases in cytoplasmic calcium, vacuolar reserve mobilization, gene transcription, and the synthesis and secretion of hydrolases. To further define elements of the Ca2+-dependent GA response machinery, we have cloned a Ca2+-dependent protein kinase (HvCDPK1) from these cells. Although expression of an inactivated (D140N) version of this kinase did not affect GA-induced gene expression or changes in cytosolic Ca2+, it did inhibit secretion, cell vacuolation, and vacuolar acidification, all responses linked to the GA response. Additionally, recombinant wild-type HvCDPK1 activated the V-type H(+)-ATPase present in isolated aleurone vacuoles. These results suggest HvCDPK1 may mediate Ca2+-dependent events of the GA response, such as control of vacuolar function, that lie downstream of transcriptional regulation.