6-Chloro-5,10-dihydro-5-[( 1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11one++ + hydrochloride (UH-AH 37) is an analog of pirenzepine that has previously been reported to interact with classical muscarinic antagonists in a competitive manner, yet its binding has also been found to be sensitive to the same epitope as is that of the allosteric ligand gallamine. The present study was carried out with wild-type and chimeric muscarinic receptors to determine whether UH-AH 37 might also have an allosteric mode of action. In assays that detect only allosteric interactions, UH-AH 37 slowed the rate of dissociation of [3H]N-methylscopolamine (NMS) from all five muscarinic receptor subtypes, with the highest apparent affinity at m2. By contrast, studies carried out under equilibrium conditions have found UH-AH 37 to have the lowest affinity for the m2 subtype. Studies with m2/m5 chimeric receptors found the allosteric potency of UH-AH 37 to be sensitive to an epitope in the seventh transmembrane domain (TM). Again, this contrasts with equilibrium studies, wherein an epitope in the sixth TM has been implicated. Simultaneous analysis of the interactions between UH-AH 37 and [3H]NMS at the m2 receptor under equilibrium and non-equilibrium conditions found that a simple allosteric model could not accommodate both sets of data. On the other hand, the model did accommodate such data for gallamine; gallamine also displays concordance in order-of-potency and epitope sensitivity between equilibrium and non-equilibrium assays. Based on these results, we conclude that UH-AH 37 interacts at the classical muscarinic binding site with high affinity and at a second (allosteric) site with lower affinity.