Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (C×36) is the most abundant isoform; however, the mechanisms underlying formation of C×36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native C×36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-C×36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of C×36-EGFP clusters on an AII cell in the KO-C×36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced - as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling C×36. We suggest that employing different gapjunction- forming mechanisms could 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.