To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.