Cultured fetal human retinal pigment epithelium (RPE) was grown on a permeable substrate and sealed in an Ussing chamber. The average electrical resistance (R) was 330 ohm-cm2, the average transepithelial voltage (Ve) was 3.0 mV (apical side positive), and the average short circuit current (Isc) was 9.1 microA/cm2. When these RPE preparations were exposed to isoproterenol (a beta-adrenergic agonist), the Isc increased by 88%, R was reduced by 6%, and Ve increased by 85%. The effect of isoproterenol was blocked by propranolol (a beta-adrenergic antagonist). When cultured human RPE was exposed to isoproterenol, intracellular cyclic adenosine monophosphate (AMP) levels rose more than threefold. The effect of isoproterenol on cyclic AMP levels was blocked by propranolol. When the cultured RPE was exposed to dibutyryl cyclic AMP, both Ve and Isc rose by 47% with a time course similar to that which occurred when the cells were exposed to isoproterenol. Preparations treated with dibutyryl cyclic AMP did not respond to subsequently applied isoproterenol. These results indicate that cultured human RPE possesses a beta-adrenergic receptor and that stimulation of this receptor produces a change in cyclic AMP concentration which affects RPE electrical activity.