A protein kinase activity associated with maize root plasma membranes was partially purified and characterized. Biochemical properties, such as calcium dependence, inhibition by calmodulin antagonists, and absence of calmodulin stimulation; indicated that the enzyme belongs to the calcium-dependent protein kinase (CDPK) family. By means of an in-gel phosphorylation assay the molecular mass of active polypeptides was determined: two bands of 55 and 51 kDa became labelled. The same proteins were also immunodecorated by monoclonal antibodies against soybean CDPK. Results from in vitro assays demonstrated that maize H+-ATPase was a suitable substrate for this protein kinase and that the phosphorylation site was located at the C-terminal domain of the enzyme. This result was confirmed by using as substrate in phosphorylation assays the isolated C-terminal domain of the H+- ATPase expressed in Escherichia coli as a glutathione-transferase fusion protein.