Verapamil, in addition to blocking calcium channels, exhibits such "non-specific" effects on myocardium as inhibition of sodium and potassium conductances and modifications of muscarinic receptor-ligand interactions. To characterize further the effects of verapamil on the cardiac muscarinic receptor, we examined the abilities of the enantiomers of verapamil to modify the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) to purified canine sarcolemmal vesicles. Membranes were incubated with [3H]QNB and various concentrations of racemic, (+)-, or (-)- verapamil (25 or 37 degrees, pH 7.4), and reactions were terminated by rapid filtration. (-)-Verapamil (Ki of 5.3 +/- 0.2 microM) was twice as potent an inhibitor of equilibrium binding as (+)-verapamil (Ki of 11.4 +/- 0.6 microM), and this effect resulted from the ability of each enantiomer to slow [3H]QNB-receptor association. This degree of stereoselectivity, albeit at nanomolar concentrations, was similar to that observed for each enantiomer to compete for the specific phenylalkylamine site in this preparation. Verapamil also inhibited [3H]QNB-receptor dissociation, but this effect required high concentrations and demonstrated stereoselectivity opposite to that observed for association. These findings support the view that verapamil interacts with two distinct sites, possibly within membrane lipid, each with a different affinity and preference for (+)- and (-)-verapamil, to modify the muscarinic receptor.