Na(+)-K(+)- ATPase alpha-subunits in basolateral membrane vesicles (BLMVs) purified from rat parotid glands were (32)P-labeled within 5 s by incubation with [gamma-(32)P]ATP at 37 degrees C in the presence of cAMP, but no labeling occurred without cAMP. Phosphorylation of Na(+)-K(+)-ATPase was associated with a decrease in its activity. This alpha-subunit phosphorylation disappeared when BLMVs were briefly incubated with cAMP and subsequent washing before the incubation with [gamma-(32)P]ATP, indicating that catalytic subunit of protein kinase A (PKA) associated to BLMVs via binding with its RII regulatory subunit anchored on the membrane. In the absence of cAMP, a PKA catalytic subunit readily reassociated with the membrane-bound RII subunit. HT-31 peptide inhibited the Na(+)-K(+)-ATPase phosphorylation by membrane-bound endogenous PKA, indicating an involvement of A-kinase anchoring protein (AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and an RII overlay assay and was coimmunoprecipitated by anti-RII antibody. These results show that Na(+)-K(+)-ATPase of rat parotid gland acinar cells is regulated in vivo by membrane-anchored PKA via AKAP rather than by free cytosolic PKA.