The Ca2+/calmodulin (CaM)-activated adenylate cyclase has been implicated as playing an important associative role in classical conditioning in both Aplysia and Drosophila. Studies of the cyclase in mammalian cerebral cortex have suggested that Ca2+/CaM sensitivity is confined to a subpopulation of total cyclase activity. We investigated the properties of cyclase from Aplysia, rat, and bovine central nervous system membranes by using CaM-Sepharose chromatography. Although only a minority of total cyclase activity bound to the CaM column, both bound and unbound fractions of cyclase from all three species showed comparable stimulation by Ca2+ in the presence of CaM. When solubilized bovine membranes were first depleted of most of their endogenous CaM by prior chromatography, binding to the CaM column was substantially increased and Ca2+ stimulation of the unbound fraction was somewhat reduced. However, this reduction in Ca2+ sensitivity resulted from the loss of Ca2+ sensitivity during prior chromatography, rather than from the more efficient separation of Ca2+-sensitive and -insensitive forms. This finding, together with the fact that we never observed any enrichment for Ca2+ sensitivity in the bound fraction over the level in the starting preparation, suggests that the vast majority of the cyclase present in solubilized central nervous system membranes is Ca2+/CaM-sensitive.