The importance of guanyl nucleotides for calmodulin stimulation of bovine cerebral cortex adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 126.96.36.199] was examined by using a partially purified calmodulin-sensitive adenylate cyclase that was resolved from calmodulin-insensitive forms of the enzyme. By using 5'-adenylyl imidodiphosphate as a substrate, in the absence of an ATP-regenerating system, it was determined that GTP is not required for calmodulin stimulation of the enzyme. Maximal activation by 5'-guanylyl imidodiphosphate (p[NH]ppG) was 5.3-fold, whereas the combination of p[NH]ppG and calmodulin stimulated the enzyme 27-fold. Although GDP inhibited p[NH]ppG stimulation of the calmodulin-sensitive adenylate cyclase, it did not affect calmodulin stimulation. In addition, calmodulin did not alter the kinetics for activation of the enzyme by p[NH]ppG. It is concluded that GTP is not required for calmodulin stimulation of brain adenylate cyclase and that calmodulin regulation of this enzyme is probably not due to effects of calmodulin on the affinity of the guanyl nucleotide regulatory complex for guanyl nucleotides.