Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular assembly.