[3H]Batrachotoxinin A benzoate ( [3H]BTX-B) binds with high affinity to sites on voltage-dependent sodium channels in a vesicular preparation from guinea pig cerebral cortex. In this preparation, local anesthetics competitively antagonize the binding of [3H]BTX-B. The potencies of some 40 classical local anesthetics and a variety of catecholamine, histamine, serotonin, adenosine, GABA, glycine, acetylcholine, and calcium antagonists, tranquilizers, antidepressants, barbiturates, anticonvulsants, steroids, vasodilators, antiinflammatories, anticoagulants, analgesics, and other agents have been determined. An excellent correlation with the known local anesthetic activity of many of these agents indicate that antagonism of binding of [3H]BTX-B binding provides a rapid, quantitative, and facile method for the screening and investigation of local anesthetic activity.