The activation of neuronal nicotinic acetylcholine receptors (nAChRs) has been implicated in the activity-dependent development and plasticity of retina and the refinement of retinal projections. Pharmacological and functional studies have also indicated that different presynaptic nAChRs can have a modulatory function in retinotectal synapses. We biochemically and pharmacologically identified the multiple nAChR subtypes expressed on retinal afferents of the superior colliculus (SC) and lateral geniculate nucleus (LGN). We found that the alpha6beta2(*) and alpha4(nonalpha6)beta2(*) nAChRs are the major receptor populations expressed in both SC and LGN. In addition, the LGN contains two minor populations of alpha2alpha6beta2(*) and alpha3beta2(*) subtypes, whereas the SC contains a relatively large population of a new native subtype, the alpha3beta2(alpha5/beta3) nAChR. This subtype binds the alpha-conotoxin MII with an affinity 50 times lower than that of the native alpha6beta2(*) subtype. Studies of tissues obtained from eye-enucleated animals allowed the identification of nAChRs expressed by retinal afferents: in SC alpha6beta2(*), alpha4alpha6beta2(*), and alpha3beta2(*) (approximately 45, 35, and 20%, respectively), in LGN, alpha4alpha6beta2(*), alpha6beta2(*), alpha4beta2(*), alpha2alpha6beta2(*), and alpha3beta2(*) (approximately 40, 30, 20, 5, and 5%, respectively). In both regions, more than 50% of nAChRs were not expressed by retinal afferents and belonged to the alpha4beta2(*) (90%) or alpha4alpha5beta2(*) (10%) subtypes. Moreover, studies of the SC tissues obtained from wild-type and alpha4, alpha6, and beta3 knockout mice confirmed and extended the data obtained in rat tissue and allowed a comprehensive dissection of the composition of nAChR subtypes present in this retinorecipient area.