A number of the currently described adenylyl cyclase species can be regulated by Ca2+ in the submicromolar concentration range in in vitro assays. The regulatory significance of these observations hinges on whether a physiological elevation in intracellular Ca2+ can regulate these cyclase activities in intact cells. However, achieving a physiological elevation in cytosolic Ca2+ is complicated by the fact that hormonal increases in cytosolic Ca2+ can be accompanied by additional effects, such as liberation of beta gamma-subunits of G-proteins and activation of protein kinase C, which can have disparate type-specific effects on cyclase activities. Therefore we have devised a strategy based on capacitative Ca2+ entry to show that, when types I and VI adenylyl cyclase are expressed in human embryonic kidney 293 cells, they are stimulated and inhibited respectively by Ca2+ entry. Blockade of Ca2+ entry by La3+ ions blocks the effects of Ca2+ entry on cyclic AMP synthesis. These studies establish that adenylyl cyclases deemed to be sensitive to Ca2+ in in vitro assays can be regulated by physiological Ca2+ entry, and therefore, such cyclases are poised to respond to changes in intracellular Ca2+ in tissues in which they are expressed.