Characterization of adenylyl cyclases has been facilitated by the isolation of cDNA clones for distinct adenylyl cyclases including the type I and type III enzymes. Expression of type I adenylyl cyclase activity in animal cells has established that this enzyme is stimulated by calmodulin and Ca2+. Type III adenylyl cyclase is enriched in olfactory neurons and is regulated by stimulatory G proteins. The sensitivity of the type III adenylyl cyclase to Ca2+ and calmodulin has not been reported. In this study, type III adenylyl cyclase was expressed in human kidney 293 cells to determine if the enzyme is stimulated by Ca2+ and calmodulin. The type III enzyme was not stimulated by Ca2+ and calmodulin in the absence of other effectors. It was, however, stimulated by Ca2+ through calmodulin when the enzyme was concomitantly activated by either GppNHp or forskolin. The concentrations of free Ca2+ for half-maximal stimulation of type I and type III adenylyl cyclases were 0.05 and 5.0 microM Ca2+, respectively. These data suggest that the type III adenylyl cyclase is stimulated by Ca2+ when the enzyme is activated by G-protein-coupled receptors and that increases in free Ca2+ accompanying receptor activation may amplify the primary cyclic AMP signal.