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To investigate the effects of nucleoside triphosphates on the activation of adenylate cyclase by choleragen and on the stability and catalytic function of the choleragen-activated enzyme, we treated samples of particulate preparation from bovine brain successively in three separate incubations with extensive washing between each step. In incubation I, choleragen and NAD were pesent to activte the adenylate cyclase. In incubation II, conditions were varied to assess enzyme stability. Finally, adenylate cyclase activity was assayed with ATP or adenylyl imidodiphosphate [App-(NH)p] as the substrate. Even when assays contained an optimal concentration of GTP, nucleoside triphosphate (plus a regenerating system) was required in incubation I for maximal choleragen activation; in order of effectiveness, GTP > ITP > ATP greater than or equal to CTP = UTP. During incubation II (at 30 degrees C), activity of the choleragen-treated fractions was essentially completely stable when 100 microM GTP (plus a regenerating system) was present. ITP and ATP were less effective. Activation produced by guanylyl imidodiphosphate was more stable than that resulting from choleragen, GTP, and NAD. After activation of membranes with choleragen, NAD, and GTP, nucleoside triphosphate plus a regenerating system (but not NAD or additional choleragen) was essential for expression of maximal activity. In order of effectiveness, GTP > ITP > ATP greater than or equal to CTP = UTP. It appears that GTP, which was effective in micromolar concentrations, plays an important role not only in the activation of adenylate cyclase by choleragen but also in the stabilization and expression of the catalytic function of the activated enzyme.