The determinants of stereospecific binding of type I antiarrhythmic drugs to specific sites associated with the sodium channel were assessed using rat cardiac myocytes. The asymmetric carbon atoms of stereoisomers may be located at two sites within type I drugs. The structure of these drugs can be schematically illustrated as Aromatic-C1-link-C2-Amine, where C1 and C2 represent potentially asymmetric carbon atoms. We used enantiomeric pairs with either C1 or C2 asymmetric carbon atoms to assess the importance of conformation at these sites to drug binding. The affinities of enantiomers of seven sodium channel blockers were measured with a radioligand binding assay using [3H]batrachotoxinin benzoate [( 3H]BTXB) and freshly isolated cardiac myocytes. The enantiomers inhibited [3H]BTXB binding in a dose-dependent manner, with a mean Hill number of 1.0 +/- 0.1. The ratios of affinities [IC50 of (+)-isomer/IC50 of (-)-isomer] were, for the C1 pairs: quinidine, 0.29; cinchonidine, 0.55; disopyramide, 1.11; RAC 109, 5.33; and for C2 pairs: flecainide, 1.03; mexiletine, 2.15; tocainide, 3.01. The stereospecific differences in drug binding suggest that the orientations of both the aromatic and the amine groups to the rest of the drug molecule are important determinants of drug binding to the cardiac sodium channel. This also suggests the presence of at least two stereospecific domains within the binding sites for type I antiarrhythmic drugs.