Na⁺-K⁺- ATPase -subunits in basolateral membrane vesicles (BLMVs) purified from rat parotid glands were ³²P-labeled within 5 s by incubation with [-³²P]ATP at 37°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 -subunit phosphorylation disappeared when BLMVs were briefly incubated with cAMP and subsequent washing before the incubation with [-³²P]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.