The ciliary body contains an epithelial bilayer consisting of an outer pigmented cell layer (PE) and an inner nonpigmented cell layer (NPE) responsible for aqueous humor secretion. Secretion may be mediated in part by cytosolic Ca2+ concentration ([Ca2+]i), but whether or how the two layers could coordinate their Ca2+ signals to regulate secretion is unclear. To investigate interactions between PE and NPE, we examined [Ca2+]i signaling in isolated intact ciliary epithelial bilayers using confocal microscopy. Phenylephrine selectively increased [Ca2+]i in PE and acetylcholine increased [Ca2+]i in NPE, but epinephrine increased [Ca2+]i in both layers. This increase spread from PE to NPE, and [Ca2+]i signaling across the bilayer remained coordinated during [Ca2+]i oscillations. All epinephrine-induced [Ca2+]i signaling was blocked by the alpha1-adrenergic antagonist prazosin, whereas signaling in the NPE but not PE was blocked by the beta-adrenergic antagonist propranolol, the gap junction blockers octanol and 18alpha-glycyrrhetinic acid, or the A kinase inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. The beta-adrenergic agonist isoproterenol failed to increase Ca2+ by itself, but isoproterenol plus phenylephrine-induced [Ca2+]i signals across the bilayer similar to those induced by epinephrine. Finally, isoproterenol increased cell-to-cell spread of lucifer yellow via gap junctions, whereas cell-to-cell spread of [Ca2+]i signals could be induced by photorelease of caged inositol 1,4,5-trisphosphate. Thus, calcium signals are coordinated in the epithelial bilayer so that adrenergic stimulation can increase [Ca2+]i in NPE, but only if NPE are primed by activation of endogenous adenylyl cyclase, whereupon they receive stimulation from adjacent PE via gap junctions. This novel interplay between endocrine and paracrine pathways may coordinate [Ca2+]i signaling across the ciliary epithelial bilayer.