The distribution of glycinergic synapses in macaque monkey retina was investigated. The monoclonal antibody (mAb2b) against the αl subunit of the glycine receptor produced a punctate immunoreactivity that was localized to synapses. In central retina about 70% of the αl subunit-containing synapses were located in strata 1 and 2 of the inner plexiform layer, about 30% were located in strata 3 and 4, and immunoreactivity was absent in stratum 5. Electron microscopy showed that the majority of the synapses in strata 1 and 2 were on cone bipolar axons. The presynaptic profile always belonged to an amacrine cell. Presynaptic and postsynaptic profiles were further characterized using double-label immunofluorescence with cell-type specific antibodies against calcium-binding proteins. An antiserum against calretinin was used to label A<doubt/>II amacrine cells and an antiserum against recoverin was used to label flat midget bipolar cells. In the outer part of the IPL, 75% of the αl-immunoreactive puncta were colocalized with calretinin-immunoreactive An processes and 61% of the αl-immunoreactive puncta were colocalized with recoverin-positive midget bipolar axons. These results suggest that the αl subunit of the glycine receptor is present at the chemical synapse made by A<doubt/>II amacrine cells with flat midget bipolar cells, thus providing a pathway for rod signals to reach midget ganglion cells.