Type V and VI mammalian adenylyl cyclases (AC5, AC6) are inhibited by Ca(2+) at both sub- and supramicromolar concentration. This inhibition may provide feedback in situations where cAMP promotes opening of Ca(2+) channels, allowing fine control of cardiac contraction and rhythmicity in cardiac tissue where AC5 and AC6 predominate. Ca(2+) inhibits the soluble AC core composed of the C1 domain of AC5 (VC1) and the C2 domain of AC2 (IIC2). As observed for holo-AC5, inhibition is biphasic, showing "high-affinity" (K(i) = approximately 0.4 microM) and "low-affinity" (K(i) = approximately 100 microM) modes of inhibition. At micromolar concentration, Ca(2+) inhibition is nonexclusive with respect to pyrophosphate (PP(i)), a noncompetitive inhibitor with respect to ATP, but at >100 microM Ca(2+), inhibition appears to be exclusive with respect to PP(i). The 3.0 A resolution structure of Galphas.GTPgammaS/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATPalphaS and 8 microM free Ca(2+) contains a single, loosely coordinated metal ion. ATP soaked into VC1:IIC2 crystals in the presence of 1.5 mM Ca(2+) is not cyclized, and two calcium ions are observed in the 2.9 A resolution structure of the complex. In both of the latter complexes VC1:IIC2 adopts the "open", catalytically inactive conformation characteristic of the apoenzyme, in contrast to the "closed", active conformation seen in the presence of ATP analogues and Mg(2+) or Mn(2+). Structures of the pyrophosphate (PP(i)) complex with 10 mM Mg(2+) (2.8 A) or 2 mM Ca(2+) (2.7 A) also adopt the open conformation, indicating that the closed to open transition occurs after cAMP release. In the latter complexes, Ca(2+) and Mg(2+) bind only to the high-affinity "B" metal site associated with substrate/product stabilization. Ca(2+) thus stabilizes the inactive conformation in both ATP- and PP(i)-bound states.