Here we provide a full kinetic characterization of the V-ATPase in microsomal preparations from the posterior gills of D. pagei acclimated to different salinities (< 0.5‰ to 21‰) for 10 days, and we examine the time course of acclimation to a single hyperosmotic challenge (21‰). Our data reveal remarkable alterations in the specific activity and kinetic properties of the enzyme in response to salinity acclimation and during the time course of exposure to 21‰, indicative of short- and long-term modulatory mechanisms of V-ATPase activity.