Vasopressin (V) causes a sustained increase in Na reabsorption and K secretion in isolated cortical collecting ducts (CCD) from rats. Because increased Na reabsorption may be associated with increased Na+-K+-ATPase activity, we investigated effects of V, given either in vivo or in vitro, on Na+-K+-ATPase activity in isolated nephron segments of rats. Na+-K+-ATPase activities were measured by coupling the hydrolysis of ATP to the production of a fluorescent nucleotide. In addition to CCD, other microdissected structures were medullary thick ascending limbs of Henle's loop, cortical thick ascending limbs of Henle's loop, and outer medullary collecting duct. To determine the time course of the response, Na+-K+-ATPase activities were measured in CCD 1 h, 3 h, 1 day, 3 days, and 7 days after intramuscular administrations of V. There was a significant increase in Na+-K+-ATPase activity in CCD after in vivo V administration for 7 days but not in any other segment. The activities increased after 3 days of administration of V. For in vitro experiments, CCD were incubated with 10(-6) M V for 1-3 h. Na+-K+-ATPase activities did not change after 1- or 3-h exposure of V in CCD in vitro. We conclude that prolonged V administration in vivo increases Na+-K+-ATPase activity in CCD. Because, in vitro exposure to V does not increase Na+-K+-ATPase activity, we conclude that rapid V-dependent increases in Na and K transport previously demonstrated in isolated perfused tubules are not dependent on a change in maximal Na+-K+-ATPase activity.