1. We have developed a plasma membrane preparation from the mucosal epithelium of rabbit gallbladder and have characterized the hormonal sensitivity of adenylate cyclase in this preparation. 2. Basal activity is low and is stimulated by GTP and GppNHp. Hormonal stimulation is largely dependent on exogenous guanine nucleotide. 3. Several prostaglandins (E1 approximately E2 greater than A1 greater than B1), vasoactive intestinal peptide and the beta-adrenergic agonist, isoproterenol, stimulate mucosal adenylate cyclase activity; a variety of peptides and neurotransmitters (secretin, cholecystokinin, arg-vasopressin, oxytocin, histamine, dopamine and serotonin) are without effect. 4. The data support the hypothesis that the inhibitory effect of prostaglandins, vasoactive intestinal peptide, and isoproterenol on gallbladder fluid absorption in certain species may be mediated by cyclic AMP. 5. The membrane preparation should be useful in further characterizing hormone receptor-transducer interactions of the gallbladder mucosal epithelium.