Electrical synapses (gap junctions) allow for rapid signal transmission between neurons. They are formed by connexins and regulated by a variety of intracellular pathways. In neurons, connexin36 (Cx36) represents the most abundant isoform. The mechanisms forming Cx36-containing electrical synapses are unknown although previous studies indicate that an intact carboxy-terminus is necessary: mice lacking the native Cx36 but expressing a carboxy-terminal fusion variant (KO-Cx36-EGFP) cannot form gap junctions in the olfactory bulb and cerebellum. We now report that neurons may harbor a different mechanism for Cx36 assembly. Using the mouse retina as a model system, we focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In KO-Cx36-EGFP mice, we demonstrate that heterocellular AII–ON cone bipolar cell gap junctions are fully functional whereas AII–AII homocellular gap junctions are not formed. Tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstructing the set of Cx36-EGFP clusters located on an AII cell in the KO-Cx36-EGFP genotype confirmed that their number but not average size is reduced, as expected for AII cells lacking a subset of electrical synapses. In conclusion, our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap junction-forming mechanisms may provide the means for a cell to regulate its gap junctions in a target cell-specific manner even if these junctions contain the same connexin.