The interaction of gallamine with muscarinic receptor subtypes was examined using radioligand binding studies. In competition studies using [3H]N-methylscopolamine [( 3H]NMS), gallamine displayed high affinity for the rat cardiac and guinea-pig uterine M2 muscarinic receptors and for the atypical muscarinic receptor present in chicken heart. Gallamine displayed low affinity for rat glandular and human 1321 N1 astrocytoma cell M3 receptors and also for the M4 receptors of NG108-15 and PC12 cells. The compound displayed intermediate affinity for M1 receptors of rat cortex labeled using [3H]pirenzepine. The interaction of gallamine with the M1 and M2 receptors appeared to be competitive at the low concentrations required to determine affinity estimates. Thus, gallamine inhibited the binding of [3H]pirenzepine to M1 receptors and [3H]NMS to M2 receptors at concentrations that were 263- and 23-fold lower, respectively, than those required to decrease radioligand dissociation kinetics. Furthermore, gallamine, at a concentration that inhibited between 63 and 71% of specific radioligand binding, had no effect on the observed rate of association of the radioligand with either the M1 or the M2 receptor. At the M3 glandular receptor, there was little separation between the concentrations of gallamine that produced inhibition of binding and those that decreased the association and dissociation rates of [3H]NMS. It is therefore difficult to determine if the inhibition of binding seen in competition studies on the M3 receptor was produced through a competitive or an allosteric mechanism. Despite its possible allosteric properties at the M3 receptor, gallamine can be used to detect heterogeneity of muscarinic receptor subtypes in several tissues and therefore represents a useful tool for defining muscarinic receptor subtypes.