Acetylcholine (ACh) in the vertebrate retina affects the response properties of many ganglion cells, including those that display directional selectivity. Three β and eight α subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been purified and antibodies have been raised against many of them. Here we describe biochemical and immunocytochemical studies of nAChRs in the rabbit retina. Radioimmunoassay and Western blot analysis demonstrated that many of the nAChRs recognized by a monoclonal antibody (mAb210) contain β2 subunits, some of which are in combination with α3 and possibly other subunits. MAb210-immunoreactive cells in the inner nuclear layer (INL) were 7-14 μm in diameter and were restricted to the innermost one or two tiers of cells, although occasional cells were found in the middle of the INL. At least 60% of the cells in the ganglion cell layer (GCL) in the visual streak displayed mAb210 immunoreactivity; these neurons ranged from 7-18 μm in diameter. The dendrites of cells in both the INL and GCL could sometimes be followed until they entered one of two dense, poorly defined, bands of processes in the inner plexiform layer (IPL) that overlap the arbors of the cholinergic starburst cells. Parvalbumin and serotonin-positive neurons did not exhibit nAChR immunoreactivity. Although the level of receptor expression appeared to be low, mAb210 immunoreactivity was observed in some of the ChAT-positive (starburst) amacrine cells.