We have characterized muscarinic cholinergic receptor sites and interaction of various modulators of intracellular Ca mobilization, such as 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, procaine and related drugs with the receptor sites, using a binding assay method with [3H]quinuclidinyl benzilate [( 3H]QNB) in guinea pig tenia cecum membrane fraction. [3H]QNB bound to a single population of sites, and the binding profile of pirenzepine suggest that the receptor sites are probably the M2 type. All the test drugs examined except for ryanodine displaced the [3H]QNB binding, and the slope factors were not different from unity. Affinities of these drugs were comparable to the concentrations usually used to modulate intracellular Ca mobilization. A Scatchard plot of [3H]QNB binding in the presence of TMB-8 or procaine showed that the apparent dissociation constant for [3H]QNB was increased without change in maximum binding. Neither TMB-8 nor procaine had any effect on the rate of dissociation of [3H]QNB from the steady-state complex. These results suggest that TMB-8 and procaine, and possibly other drugs, may interact directly with the muscarinic cholinergic M2 receptor sites in a competitive manner. In contrast, ryanodine had no effect on the [3H]QNB binding up to at least 100 microM. No interaction of ryanodine with M2 receptor sites lends a further support that the drug could be a useful probe for studying intracellular Ca mobilization.