To identify the binding domain for brevetoxins, a family of lipid-soluble neurotoxins acting at Na+ channel receptor site 5, purified and reconstituted rat brain Na+ channels were photolabeled with p-azidobenzoyl tritium-labeled brevetoxin, and the labeled peptides were identified. A radiolabeled band with an apparent molecular mass of 250 kDa corresponding to the Na+ channel alpha-subunit was revealed using both gel slicing and fluorography techniques. Regions of the alpha-subunit specifically photolabeled by this ligand were then identified by antibody mapping of proteolytic fragments. Even after extensive proteolysis, anti-peptide antibodies recognizing amino acid sequences within or adjacent to Na+ channel transmembrane segments IS6 and IVS5 were each able to immunoprecipitate up to 40% of the labeled peptides. A more extensive tryptic digest was obtained with a preparation in which the brevetoxin photolabel was incorporated into the alpha-subunit of purified Na+ channel in detergent solution. The identification of a specifically immunoprecipitated 6-kDa peptide containing transmembrane segment S6 from domain I restricted the labeled peptide fragment to residues Thr-400 to Lys-443 if tryptic digestion was complete or Ala-396 to Lys-455 if tryptic cleavage was incomplete. Similarly, the identification of a specifically immunoprecipitated 6-kDa peptide from domain IV restricted the labeled peptide to residues Glu-1738 to Lys-1785 or Glu-1738 to Lys-1793 on the extracellular side of transmembrane segment S5. These results provide direct evidence for close association of transmembrane segments IS6 and IVS5 in the native conformation of the Na+ channel alpha-subunit and implicate their region of interaction as an important component of the brevetoxin receptor site.