The distribution of glycinergic synapses in macaque monkey retina was investigated. The monoclonal antibody (mAb2b) against the alpha 1 subunit of the glycine receptor produced a punctate immunoreactivity that was localized to synapses. In central retina about 70% of the alpha 1 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 AII 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 alpha 1-immunoreactive puncta were colocalized with calretinin-immunoreactive AII processes and 61% of the alpha 1-immunoreactive puncta were colocalized with recoverin-positive midget bipolar axons. These results suggest that the alpha 1 subunit of the glycine receptor is present at the chemical synapse made by AII amacrine cells with flat midget bipolar cells, thus providing a pathway for rod signals to reach midget ganglion cells.