The development of muscarinic acetylcholine receptor protein in the ferret retina was studied using biochemical, autoradiographic, and light and electron microscopic immunohistochemical techniques. The development of retinal muscarinic cholinergic receptor proteins involves transient shifts in their number and distribution, as well as changes in the relative abundance of two molecular weight variants. Receptor binding assays demonstrate changes in the number and affinity of retinal binding sites for the muscarinic cholinergic ligand [3H]quinuclidinylbenzilate ([3H]QNB). Light microscopic immunohistochemical studies reveal the presence of muscarinic acetylcholine receptor-like (mAChR-like) immunoreactivity in the adult inner plexiform layer. During development, the mAChR-like immunoreactivity appears in a number of other retinal layers. Electron microscopic immunohistochemical studies indicate that muscarinic acetylcholine receptor-like immunoreactivity is found at amacrine-amacrine cell contacts. Both autoradiographic and gel slice electrophoretic studies were carried out after labeling of developing and adult retinal muscarinic receptors with [3H]propylbenzilylcholine mustard ([3H]propylbenzilylcholine mustard ([3H]PrBCM), which irreversibly labels the muscarinic acetylcholine receptor. Polyacrylamide gel electrophoresis under reducing, denaturing conditions resolved two peaks of radioactivity corresponding to [3H]PrBCM-labeled protein; both were eliminated by pre- and co-incubation of labeled adult retinas with excess atropine. Combined with the results of earlier studies, these observations suggest that the subtypes, number and distribution of muscarinic receptor proteins changes during retinal synaptogenesis.