The distribution of muscarinic and nicotinic cholinergic binding sites in the cat nucleus tractus solitarii was studied by the technique of in vitro autoradiography. Using the antagonist [3H]quinuclidinyl benzilate, muscarinic binding sites were differentially located in subdivisions of the nucleus tractus solitarii. The majority of muscarinic binding sites were located predominantly in the caudal half of the nucleus, reaching their greatest amounts at the mid levels of the nucleus tractus solitarii. The medial, dorsolateral, intermediate, and interstitial subdivisions contained the highest densities of quinuclidinyl benzilate binding sites. Nicotinic cholinergic binding sites, using [3H]nicotine and [125I]alpha‐bungarotoxin, had unique patterns of distribution. With [3H]nicotine the majority of binding sites were located in rostral levels of the nucleus with very few binding sites present in the caudal half. In contrast, [125I]alpha‐bungarotoxin binding sites were present mainly in subdivisions located in the caudal half of the nucleus, i.e., commissural, ventrolateral, dorsolateral, medial, and intermediate subdivisions, and dropped off precipitously at more rostral levels. The differential distribution of [3H]nicotine and [125I]alpha‐bungarotoxin suggests the two ligands may be labeling different types of nicotinic binding sites in the nucleus tractus solitarii. The unique distribution of muscarinic and nicotinic cholinergic binding sites in the various subdivisions of the nucleus solitarii suggests that muscarinic and nicotine mechanisms may play an active role in the regulation of the diverse autonomic functions at the level of the nucleus tractus solitarii. © 1993 Wiley‐Liss, Inc.