The intrinsic cAMP-dependent protein kinase activity of highly purified cardiac sarcolemmal vesicles was characterized. The sarcolemmal protein kinase was specifically activated by cAMP. Binding of cAMP to the kinase was saturable and occurred exclusively to a protein of Mr = 55,000 intrinsic to the vesicles. This binding of cAMP to the sarcolemmal vesicles caused a selective release of catalytic activity from the membranes, which was capable of phosphorylating several endogenous sarcolemmal substrates as well as one additional substrate, which was also identified in purified vesicles of cardiac sarcoplasmic reticulum. Unmasking experiments conducted with the ionophore alamethicin demonstrated that the protein kinase activity and its endogenous sarcolemmal substrates were localized on the inner, cytoplasmic surfaces of the vesicles, and, furthermore, suggested that at least 75% of the vesicles were right side out. The major protein substrates phosphorylated in the sarcolemmal fraction exhibited apparent molecular weights of 21,000 and 8,000, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heating the membranes in the presence of sodium dodecyl sulfate prior to electrophoresis completely converted the 21,000-dalton substrate into the form of higher mobility, suggesting that the two substrates were, in fact, identical proteins. This was supported by the observation that both substrates exhibited identical pI values of approximately 6.7. Although present in the sarcolemmal fraction, these two substrates were not localized exclusively to sarcolemmal membranes. The same two substrates were present in 3-fold higher content in purified cardiac sarcoplasmic reticulum vesicles. Moreover, although phosphorylation of all other sarcolemmal proteins in right side out vesicles by exogenously added protein kinase was increased 4-fold or greater by alamethicin, phosphorylation of the substrates of Mr = 21,000 and 8,000 was not altered appreciably by the ionophore. The results suggest that these two major substrates identified in the sarcolemmal preparations are not intrinsic sarcolemmal proteins.