Frog liver adenylate cyclase was characterized with respect to divalent cation interaction and hormonally stimulated activities. The enzyme catalyzed the synthesis of cyclic [32P]3',5'-AMP from alpha-32P-labeled ATP. The activity of the enzyme was linear with time and protein concentration. The Km for ATP was 0.5 mM, in the presence or absence of stimulators. The temperature optimum was 25 degrees. GTP (10(-4) M) increased the stimulation of adenylate cyclase by epinephrine. Similar activities were obtained using 5 mM Mg2+ or Mn2+. At higher concentrations, both ions inhibited epinephrine-stimulated, but not basal or fluoride-stimulated activities. Approximately equivalent hormonal stimulation was obtained with maximal stimulating concentrations of epinephrine, isoproterenol, glucagon, and prostaglandin E1. Norepinephrine was less stimulatory. Only catecholamine-stimulated activities were inhibited by propranolol (10(-5) M). The data suggest that catecholamines stimulate frog liver adenylate cyclase through interactions with beta adrenergic receptors. The adenylate cyclase in frog liver differs from its mammalian counterpart in its response to temperature and maximally stimulatory concentrations of hormones.