Batrachotoxinin-A 20-alpha-benzoate (BTX-B), an analog of the potent depolarizing agent batrachotoxin (BTX), was prepared by selective esterification of naturally occurring batrachotoxin-A with benzoic acid. BTX-B depolarized rat phrenic nerve-diaphragm preparations with a time course and concentration dependence virtually indistinguishable from that of BTX. A specific, saturable component of equilibrium binding of [3H]BTX-B to mouse cerebral cortex homogenates was measured, described by an equilibrium dissociation constant of 0.7 microM and a maximum number of binding sites of 90 pmol per gram of tissue (wet weight). Specific binding is inhibited by BTX and other BTX analogs, veratridine, and grayanotoxin but is unaffected by tetrodotoxin and cevine. Under conditions of this assay, neither crude Leiurus quinquestriatus scorpion venom nor purified sea anemone toxin have any effect of specific binding. The data support the conclusion that BTX-B interacts with a recognition site associated with voltage sensitive sodium channels which is identical to the recognition site for BTX.