Physiological studies have demonstrated the existence of direct intercellular communication, presumably mediated by gap junctions, both between neurons and between glial cells in the vertebrate retina. We localized gap junctions in the retinas of rat, goldfish, and mudpuppy by using antisera directed against proteins that make up the connexon channels in two tissues from which connexins have been isolated: liver (connexin 32; CX32) and heart (connexin 43; CX43). Although the antiserum against CX32 stained liver gap junctions, it did not reveal any staining in rat or goldfish retina. The antiserum against CX43 stained gap junctions associated with the intercalated disk in rat heart and also stained gap junctions between pigment epithelium cells in rat, goldfish, and mudpuppy retina. Anti-CX43 also stained gap junctions between Müller cells in goldfish and mudpuppy retina but not in rat retina. Intracellular injections of the tracer Neurobiotin into Müller cells in the mudpuppy retina revealed that these glial cells are extensively tracer coupled. Staining with the tracer formed a syncytium of thin processes surrounding every neuron from the outer limiting membrane to the inner limiting membrane. Confocal microscopy demonstrated that the Müller cells were in close apposition with one another at every level of the retina. However, CX43 immunoreactivity was heaviest at the outer limiting membrane, where the apical processes of Müller cells are located. Some anti-CX43 staining was observed at the level of the outer nuclear layer and the inner plexiform layer but not in the ganglion cell layer or at the Müller cell end feet forming the inner limiting membrane.