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 ␣62* and ␣4(non␣6)2* nAChRs are the major receptor populations expressed in both SC and LGN. In addition, the LGN contains two minor populations of ␣2␣62* and ␣32* subtypes, whereas the SC contains a relatively large population of a new native subtype, the ␣32(␣5/3) nAChR. This subtype binds the ␣-conotoxin MII with an affinity 50 times lower than that of the native ␣62* subtype. Studies of tissues obtained from eyeenucleated animals allowed the identification of nAChRs expressed by retinal afferents: in SC ␣62*, ␣4␣62*, and ␣32* (approximately 45, 35, and 20%, respectively), in LGN, ␣4␣62*, ␣62*, ␣42*, ␣2␣62*, and ␣32* (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 ␣42* (90%) or ␣4␣52* (10%) subtypes. Moreover, studies of the SC tissues obtained from wild-type and ␣4, ␣6, and 3 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.Neuronal nicotinic acetylcholine receptors (nAChRs) are highly expressed in the visual system, where they play important roles, not only in the retina itself but also in retinal target tissues (for review, see Feller, 2002). nAChRs are a family of heterogeneous, pentameric receptor complexes consisting of different ␣ and  subunits whose opening is physiologically controlled by the neurotransmitter acetylcholine (for review, see Gotti and Clementi, 2004). So far, 12 different subunits have been identified (␣2-␣10, 2-4) whose various