Binding of 3H-saxitoxin to Na+ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1 nM; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30 nM. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K+ concentrations, or by activation of the Na+ channels with batrachotoxin plus a purified polypeptide toxin from the scorpion Leiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70--80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60--70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2 nM. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0 degrees of 0.1 min-1 for membrane bound and 0.2 min-1 for detergent-solubilized binding sites. The measured association rate constant was 6 X 10(8) M-1 min-1 at 0 degrees for membrane-bound saxitoxin binding sites.