Allosteric regulation of [3H]N-methylscopolamine [( 3H]NMS) and [3H]quinuclidinyl benzilate [( 3H]QNB) dissociation from the m1-m5 muscarinic receptor subtypes was examined in transfected CHO-K1 cells. Half-times of dissociation of [3H]NMS from cell membranes (at 23 degrees) ranged from less than 5 min for the m2 subtype to more than 60 min for the m5 subtype. For [3H]QNB, half-times (at 37 degrees) ranged from 1 hr (m2) to almost 4 hr (m3). The presence of gallamine slowed the dissociation of [3H]NMS from all of the subtypes, with an order of potency of m2 greater than m4 greater than m1 greater than m3 greater than m5. Dissociation of [3H]QNB from m1 and m2 receptors was modulated by gallamine in the biphasic manner that we have described previously for cardiac receptors; that is, low concentrations (1-10 microM) of gallamine accelerated dissociation, while 1 mM gallamine slowed it. Verapamil slowed the dissociation of [3H]-QNB from the m2 receptor in a monophasic manner, while the action of d-tubocurarine was qualitatively similar to that of gallamine. The potency of gallamine in allosterically regulating the m2 receptor was inversely related to ionic strength. Inactivation of pertussis toxin-sensitive G proteins abolished the ability of guanine nucleotides to regulate agonist affinity at the m2 receptor, but had no effect on allosteric regulation of the m2 receptor. These findings indicate that susceptibility to allosteric regulation varies in a complex way across muscarinic receptor subtypes and according to the choice of ligand.