Type III adenylyl cyclase is stimulated by β-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca²⁺ and calmodulin. However, the enzyme is stimulated by Ca²⁺ and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide stimulatory protein G_s (Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31, 6492-6498). Here, we examined regulation of type III adenylyl cyclase by G_s-coupled receptors and intracellular Ca²⁺in vivo. Surprisingly, intracellular Ca²⁺ inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca²⁺, which stimulated type I adenylyl cyclase, inhibited glucagon- or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca²⁺ was not mediated by G_i, cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca²⁺ inhibition of the enzyme, and coexpression of constitutively activated CaM kinase II completely inhibited isoproterenol-stimulated type III adenylyl cyclase activity. We propose that Ca²⁺ inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.