Glycine receptors (GlyRs) and their role in retinal circuitry were analyzed immunocytochemically in wild‐type and GlyR α3 subunit–deficient (Glra3^−/−) mouse retinae. GlyRs are localized in the inner plexiform layer in brightly fluorescent puncta, which are likely to represent postsynaptically clustered GlyRs. Approximately one third of the clusters were found to contain the α1 subunit, and half possessed the α3 subunit. However, these two GlyR isoforms were localized at different glycinergic synapses. In the Glra3^−/− mouse, α3 subunit clusters were completely eliminated, although the total number of GlyR clusters was only slightly reduced. This finding indicates that other GlyR subunits (such as α2 or α4) may have compensated for the loss of the α3 subunit. Characteristic expression patterns of the α1 and α3 subunits within the synaptic circuits of the retina were revealed by double labeling sections for GlyRs and markers that define specific retinal neurons. The α1 subunit mediates signal transfer in the rod pathway between AII amacrine cells and OFF‐cone bipolar cells. In contrast, the α3 subunit appears to be predominantly involved with the cone pathways. Thus, expression of different GlyR α subunit genes correlates with anatomically defined connectivities. J. Comp. Neurol. 465:524–539, 2003. © 2003 Wiley‐Liss, Inc.