Immunohistochemical and in situ hybridization techniques were used to localize the beta 4 subunit of the neuronal nicotinic acetylcholine receptors (nAChRs) in the ground squirrel retina. The beta 4 nAChR subunit was detected in both transverse and horizontal sections of the retina using a subunit-specific antiserum and the avidin-biotin complex technique. Two bands of labeled processes were seen in the inner plexiform layer, corresponding approximately to the laminae where the cholinergic cells arborize. Labeled cells were found in the ganglion cell layer and the inner third of the inner nuclear layer. The cells in the ganglion cell layer were medium- to large-sized and were frequently observed to give rise to axon-like processes. Most of the labeled neurons in the inner nuclear layer were small presumptive amacrine cells, but a few medium-to-large cells were also labeled. These could constitute a different class of amacrine cells or displaced ganglion cells. The latter possibility is supported by the existence of nAChR-containing displaced ganglion cells in the avian retina. In situ hybridization with a 35S-labeled cRNA probe revealed the expression of mRNA coding for the nAChR beta 4 subunit in the ganglion cell layer and the inner third of the inner nuclear layer. This finding confirmed the immunohistochemical data of the cellular localization of beta 4 nAChR subunit. These results indicate that the beta 4 nAChR subunit is expressed by specific subtypes of neurons on the ground squirrel retina.(ABSTRACT TRUNCATED AT 250 WORDS)