Secretory activity in blowfly salivary glands is activated by the hormone serotonin. We have investigated the distribution and activity of two cation pumps that are possibly involved with transepithelial ion transport, i.e. Na(+)/K(+)-ATPase and vacuolar-type H(+)-ATPase (V-ATPase). By immunofluorescence labelling of secretory cells, Na(+)/K(+)-ATPase was localized on the basolateral plasma membrane and V-ATPase on the highly folded apical membrane. Activities of both ATPases were probed in salivary gland homogenates by applying specific inhibitors for these ion pumps, namely ouabain and bafilomycin A(1). In control glands, bafilomycin-A(1)-sensitive V-ATPase activity and ouabain-sensitive Na(+)/K(+)-ATPase activity accounted for 36% and 19%, respectively, of the total ATPase activity. V-ATPase activity increased approximately twofold after stimulation with serotonin, whereas Na(+)/K(+)-ATPase activity was not significantly affected. Biochemical assays provided evidence that the serotonin-induced activation of V-ATPase activity was accompanied by a recruitment of peripheral V(1) subunits from the cytosol to the plasma membrane, indicative of the assembly of V(0)V(1) holoenzymes. These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical "potassium pump" that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K(+)/nH(+) antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V(0)V(1) holoenzymes might constitute a regulatory mechanism for the control of pump activity.