Apical and basal membrane fractions from Locusta Malpighian tubules were prepared and were characterized by marker enzyme analysis. The apical membranes contained an azide- and orthovanadate-insensitive ATPase activity that was inhibited by bafilomycin A1 (IC50 = 0.44 nM) and NEM (IC50 = 2.15 microM), and thus was characterized as putative V-type ATPase. The enzyme was stimulated by a variety of monovalent cations (Tris > K = Na > choline > Li = Rb) maximal stimulation occurring at 30-40 mM. It was also stimulated by a variety of monovalent anions (maximal activation 30-40 mM), but was strongly inhibited by nitrate and thiocyanate. SDS-PAGE separation of proteins present in the various membrane fractions was carried out. The apical membrane fraction alone contained a 28 kDa protein band that bound a monoclonal antibody specific for a 28 kDa peptide which was a component of the V-type ATPase from midgut of Manduca sexta and, in native gels, possessed ATPase activity which was also sensitive to both bafilomycin and NEM but not to azide or orthovanadate. Binding of the fluorescent monoclonal antibody was located at the apical boundary of the tubule cells. It was concluded that a V-type ATPase is present at the apical surface of Locusta Malpighian tubule cells and that it is involved in their secretory functioning.