The effect of vanadate, a potent inhibitor of Na-K-ATPase, on the hydroosmotic response to vasopressin (AVP) and transepithelial voltage (Vt) in cortical collecting tubules was examined. At 37 degrees C, exposure of collecting tubules to bath vanadate (10(-4) M) for 30 min inhibited the increase in hydraulic water permeability (Lp) in response to AVP or 8-bromo-cyclic adenosine monophosphate by 68 and 76%, respectively. When vanadate was present only in the lumen no inhibition of the AVP response was observed. Incubation of tubules with ouabain (10(-5) M) for 30 min inhibited the AVP-induced increase in Lp to the same extent as vanadate. At 25 degrees C, vanadate inhibited the increase in Lp by AVP if added before but not after the hormone. Addition of vanadate to the bath caused a rapid decrease in the lumen-negative Vt that is consistent with Na-K-ATPase inhibition. Luminal vanadate also inhibited Vt but the rate of decrease of Vt was much slower than in the presence of bath vanadate. We conclude that vanadate inhibits the development but not the maintenance of the AVP-induced increase in water permeability in the collecting tubule. Since the effect of ouabain was similar to that of vanadate, the results suggest that inhibition of Na-K-ATPase directly or indirectly interferes with the initiation of the AVP-induced increase in luminal membrane water permeability at a site distal to cAMP formation.