Adenylate cyclase in rat adipocyte membranes was inactivated as a result of treatment with sulfhydryl oxidants or with p-chloromercuribenzoate as well as by S-alkylating agents. The inhibition of the basal and isoproterenol- or glucagon-stimulated enzyme activity by the oxidants or the mercurial could be reversed by adding thiols to the isolated membranes. The activity of the enzyme paralleled the cellular glutathione (GSH) content. Lowering of intracellular glutathione by incubating the cells with specific reactants resulted in the inhibition of both basal and hormone-stimulated adenylate cyclase activity in the isolated membranes. Activity could be partly restored by supplying glucose to the incubation medium of intact cells. The fluoride-stimulated adenylate cyclase was also inhibited by the oxidants or the sulfhydryl inhibitors. The results suggest that adenylate cyclase may be partly regulated by oxidation-reduction. Thus, a direct relationship between both basal and hormone-stimulated adenylate cyclase activity and the cellular redox potential, determined by the cellular level of reduced glutathione, may be ascribed to the protection of the catalytic -SH groups of the enzyme from oxidative or peroxidative reactions and maintenance of the redox optimum for the reaction.