The mechanism by which the type Iα regulatory subunit (RIα) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIα is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIα gene product. A single RIα phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIα. Because both R subunit preparations were 30–40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIα. Mass spectrometry also indicated that membrane-associated RIα had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIα were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIα strongly suggests an interaction between RIα and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIα membrane association in the heart.