Prior studies have suggested the presence of muscarinic acetylcholine receptor (MAChR) subtypes within the mammalian central nervous system on the bases of functional ligand binding or molecular biologic evidence. Autoradiographic differentiation of MAChR subtypes in ligand binding assays has previously relied on the use of agonists, results of homogenate binding assays to determine relative subtype binding affinities or both. In the present study, the binding of [3H]scopolamine to intact slide-mounted tissue sections is characterized. The rapid binding kinetics of the ligand permit autoradiographic saturation experiments. Autoradiographic competition assays utilizing [3H]scopolamine and the unlabeled subtype-selective ligands pirenzepine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5,11,-dihydro-6H-pyr ido (2,3-b)(1,4)benzodiazepin-6-on (AF-DX 116) and 4-diphenylacetoxy-N-methylpiperidine methiodide reveal evidence for the presence of multiple MAChR subtypes distributed heterogeneously throughout the brain. High-affinity pirenzepine MAChR (putative M1 subtype) predominate in the telencephalon. High-affinity AF-DX 116 MAChR (putative M2 subtype) are widely distributed, but are quantitatively minor populations, with the exception of motor cranial nerve nuclei and the basal pons, where they represent the dominant MAChR fractions. Evidence for relative enrichment of M3 receptors was obtained in the thalamus, the superficial layer of the superior colliculus, the periqueductal region, the substantia nigra pars reticulata and the pons. The autoradiographic assays developed in this work may assist in defining altered receptor populations arising in pathologic conditions or resulting from drug therapy.