TMB-8, a putative inhibitor of intracellular calcium mobilization, prevents the binding of the muscarinic ligand N-[3H]methylscopolamine. The inhibition was observed in four tissues from guinea pig; cortex, heart, pancreas, and ileum, representing M1, cardiac M2, glandular M2, and heterogeneous M2 subtypes of muscarinic receptors, respectively. The Ki values for all four tissues were approx. 4 microM. However, dissociation kinetics revealed that TMB-8 interacted with an allosteric site of three muscarinic receptor subtypes but not the subtype from pancreas. These results indicate that TMB-8 interacts with muscarinic receptors, and therefore would disrupt calcium mobilization or any second messenger system coupled to these receptors.