The mammalian adenylyl cyclase is a membrane-bound enzyme that is predicted to have 12 trans-membrane spans. Between membrane spans 6 and 7 there is a large cytoplasmic loop, which, along with the C-terminal tail, makes up the catalytic site of the enzyme. Crystal structures of these soluble cytoplasmic domains have identified the regions that are involved in interactions with each other. The functional consequences of these interactions in the full-length membrane-embedded enzymes have not been established. In this study, we analyzed the role of various interaction regions within the central cytoplasmic loop (C1) and the C-terminal tail (C2) on basal, Gαs-, forskolin-, and Mn2+-stimulated activities of adenylyl cyclases 2 and 6 (AC2 and AC6). We tested synthetic peptides encoding the different interface surfaces of both the C1 and C2 domain on different activities of membrane-bound AC2 and AC6 expressed in insect cells. We found the C1-α2-β2-β3 and C2-β2′-β3′ regions to be involved in stimulation by Gαs and forskolin but not in the basal or Mn2+-stimulated activities. Both the C1-β4-β5-α4 region and the C2-α3′-β4′ region play a role in the Gαs- and forskolin-stimulated activities as well as in basal activity, because the peptides encoding these regions inhibit basal activity by 30%. In contrast, the C2-α2′ region peptide inhibits both basal and Mn 2+-stimulated activity by >50%. These results suggest that the different stimulated activities may involve distinct interface interactions in the intact enzyme and, consequently, the distinct mechanisms by which Mn 2+-activates the enzyme as compared with Gαs and forskolin, leading to the possibility that the full-length adenylyl cyclase may have multiple catalytically competent configurations.