The binding of selective muscarinic receptor antagonists to regions of rat brain was examined through quantitative autoradiographic techniques. 5,11-Dihydro-11-[(4-methyl-1-piperazinyl)acetyl]-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one [pirenzepine (compound I)] and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one [AF-DX 116 (compound II)] were chosen on the basis of their selectivity for M1 and M2 muscarinic receptors, respectively, and similarities in chemical structure. Pirenzepine displayed a higher potency than II for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain sections. Scatchard analyses of binding to brain sections revealed heterogeneous binding profiles for both antagonists, suggesting the presence of multiple receptor binding sites. Quantitative autoradiographic techniques were utilized in regional analyses of antagonist binding. Pirenzepine displayed the highest affinity for hippocampal, striatal, and amygdaloid muscarinic receptors (IC50 values less than 0.4 microM), with a slightly lower affinity for cortical receptors (IC50 values between 0.4 and 0.8 microM). Pirenzepine displayed the lowest affinity for thalamic and brainstem regions with IC50 values generally greater than 1.0 microM. In contrast, II bound with higher affinity to muscarinic receptors in brainstem, cerebellar, and hypothalamic nuclei (IC50 values less than 0.5 microM) than to receptors in thalamic nuclei (IC50 values between 0.5 and 2.0 microM). Binding sites with the lowest affinity for II were found in cortical, striatal, and hippocampal regions (IC50 values greater than 2.0 microM). The binding profiles of the two selective muscarinic antagonists reveal the complexity and diversity of muscarinic receptor subtypes throughout the brain. The data provide a basis for identifying muscarinic receptor subtypes (as defined through cloning procedures) with selective ligands. Minimum-energy conformations of pirenzepine and II were calculated by using the program MacroModel (version 2.0). Pirenzepine displayed three energy minima, differing in the relative position of the piperazine ring with respect to the tricyclic system. In contrast, the (diethylamino)methyl substituent on the piperidine ring conferred a much larger set of minimum-energy conformations on II. It is suggested that the greater conformational flexibility of the side chain allows II to achieve a conformation inaccessible to pirenzepine, which allows it to bind preferentially to M2 receptors.