Diltiazem, a benzothiazepine Ca2+ antagonist, has four stereoisomeric forms: d- and l-isomers of the cis and trans forms. All four isomers were shown to completely inhibit the binding of d-cis-[3H]diltiazem to rabbit T-tubule Ca2+ channels. The potency of the inhibition was in the order: d-cis greater than l-cis greater than d-trans = l-trans. The Hill slope for each inhibition was close to unity. The l-cis, d-trans and l-trans isomers had no effect on dissociation of the d-cis[3H]diltiazem-benzothiazepine receptor complex. These results indicate that all four isomers bind to benzothiazepine receptors. Furthermore, all of the isomers modulated (+)-[3H]PN200-110 binding. At 37 degrees C, only the d-cis isomer stimulated the binding, whereas the others showed inhibition. At 2 degrees C, all of them inhibited the binding. Both trans isomers exerted virtually the same, weak effects on the binding. It is concluded that the effects of diltiazem on radioligand binding to Ca2+ channels are highly stereospecific for the d-cis isomer.