Alpha-ganglion cells are present in all vertebrate retinae and are subdivided into ON and OFF types according to their level of dendritic ramification within the inner plexiform layer. They have large dendritic fields and usually a good responsiveness to moving stimuli. They were the first ganglion cells in which tracer coupling was observed, suggesting the presence of gap junctions composed of unknown connexins. Here we show that ON-alpha-ganglion cells in the mouse retina are coupled to amacrine cells, whereas OFF-alpha-ganglion cells are coupled to other OFF-alpha-ganglion cells and to amacrine cells. These tracer coupling patterns were completely absent in mice deficient in connexin36 (Cx36). The expression of Cx36 protein in alpha-ganglion cells but not in coupled amacrine cells was confirmed in mice in which the Cx36 coding DNA was replaced by the lacZ reporter gene. The dendritic localization and the distribution pattern of Cx36 patches, analyzed in mice in which the enhanced green fluorescent protein (EGFP) was linked to the C-terminal region of the Cx36 protein, revealed a rather small number of fluorescent plaques and different patterns for ON- and OFF-alpha-ganglion cells. Furthermore, tracer coupling between OFF-alpha-ganglion cells could be inhibited by quinine, a gap junctional blocker with a slight preference for gap junctions formed by Cx36. These data strongly suggest that Cx36 gap junction channels are functional not only in interneurons but also in output neurons of the retina and are responsible for distinct coupling patterns of ganglion cells.