A clonal rat sympathetic nerve cell line, PC12, binds iodinated alpha-bungarotoxin. The binding is saturable and is inhibited by a variety of cholinergic agonists and antagonists. The pseudo-first order rate constant for binding is 2.1 x 10(5) M-1 S-1 at 22 degrees. In contrast to the alpha-bungarotoxin binding reaction found with muscle, the binding to PC12 is reversible with a first order rate constant of 4.9 x 10(-5) S-1 at 37 degrees. Toxin binds to an integral membrane component which sediments in sucrose gradients containing Triton X-100 with an apparent sedimentation coefficient of 10.5 S. The nicotinic acetylcholine receptor of PC12 was assayed by determining the agonist-induced increase in permeability to sodium ions. Using this assay, we determined the apparent binding constants for a variety of cholinergic ligands and found no correlation between their ability to affect cholinergic function and to inhibit binding of alpha-bungarotoxin. Therefore, the site at which cholinergic ligands affect receptor function is different than the site at which cholinergic ligands inhibit toxin binding.