Adenylate cyclase in particulate fractions from rat adrenal glands is subject to regulation by purine nucleotides, particularly guanine nucleotides. While GTP activates the enzyme, this effect is not evident in all particulate fractions. Following dialysis of the refractory fractions activation by GTP is observed, an indication that endogenous nucleotides may obscure the effects of added GTP. The analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p gives considerable more activity than does GTP. GDP, on the other hand, is inhibitory, an effect revealed only in the absence of a nucleotide-regenerating solution. GDP blocks the action of both GTP and Gpp(NH)p. These results show that the gamma-phosphate of the nucleotide is required for but need not be metabolized in the activation process. At low substrate concentration (0.1 mM ATP or adenyl-5'-yl imidodiphosphate) stimulation of the enzyme by ACTH occurs only in the presence of added guanine nucleotide (GTP or Gpp(NH)p); the hormone and nucleotide act synergistically. While both GTP and Gpp(NH)p inhibit fluoride-stimulated activity, the level of fluoride required to demonstrate such inhibition appears not to be related to the level of fluoride required for activation of the enzyme. In the presence of GTP, or GTP plus ACTH, the enzyme exhibits normal Michaelis-Menten kinetics with respect to substrate utilization (K-m equal to 0.16 mM). In the activated state, produced with ACTH plus GTP, the enzyme is less susceptible to inhibition by a species of ATP uncomplexed with Mg2+, but is more susceptible to inhibition by Mg2+. These results demonstrate that fundamental differences exist between different states of the adenylate cyclase. The difficulties in describing kinetically the regulation of adenylate cyclase systems in view of the multiple actions of nucleotides and magnesium are discussed.