The capacities of the alpha subunits of pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) to inhibit different isoforms of mammalian adenylyl cyclases were assessed. Membranes from Sf9 cells infected with recombinant baculoviruses encoding either type I, II, V, or VI adenylyl cyclase were reconstituted with purified G protein subunits. Types V and VI adenylyl cyclase are most sensitive to inhibition by Gi alpha 1, Gi alpha 2, and Gi alpha 3; type I adenylyl cyclase can be inhibited by these three Gi alpha proteins and by G(o) alpha as well. Type II adenylyl cyclase appears to be immune to inhibition by these proteins. Examination of the effects of native and mutant Gi alpha proteins, as well as analysis of competition for binding of Gs alpha to adenylyl cyclases, indicate that at least certain adenylyl cyclases have independent sites for interaction with Gs alpha (site 1, stimulatory) and Gi alpha (site 2, inhibitory). High concentrations of Gi alpha can interact with site 1 on types I and II adenylyl cyclase and activate the enzymes. Types I and II adenylyl cyclase also appear to have independent sites for interaction with G protein beta gamma subunits. The type I enzyme is strongly inhibited, while type II adenylyl cyclase is activated if Gs alpha is also present.