Four monoclonal antibodies, raised against the 115-kDa adenylyl cyclase from bovine brain [Pfeuffer, E. et al. (1985) EMBO J. 4, 3675-3679] have been selected and designated BBC-1 to BBC-4. BBC-1 and BBC-3 are highly specific for the 115-kDa enzyme from bovine brain. The two other antibodies, BBC-2 and BBC-4, recognize an additional 150-kDa adenylyl cyclase in bovine brain, but also in brain tissue from other species. In membranes from lung and myocardium (bovine and rabbit) only the 150-kDa species is detected by the crossreacting antibodies BBC-2 and BBC-4. The two adenylyl cyclases from brain can be separated by calmodulin-Sepharose: only the enzyme of 115 kDa but not that of 150 kDa was retained by the affinity resin and could be stimulated by Ca2+/calmodulin. The data obtained with these antibodies of defined specificity provide for the first time direct evidence for the presence of two distinct adenylyl cyclase species in brain tissue.