Sympathetic neurons receive direct synaptic input from cholinergic terminal boutons of preganglionic nerve fibers. The distribution of acetylcholine receptors at these synapses is not precisely known. This study shows that alpha-bungarotoxin, which binds specifically to nicotinic receptors on skeletal muscle, also may be useful for localizing postsynaptic nicotinic receptors on principal neurons in the paravertebral sympathetic ganglia of the bullfrog. alpha-Bungarotoxin (1-5 microM) produces a block of nicotinic (fast) excitatory postsynaptic potentials that is fully reversed after 5-8 hr of washing. Dihydro-beta-erythroidine, a nicotinic antagonist, reduces the half-time of recovery from the toxin block to one-third of the control value, presumably by competing for the same receptor sites. Furthermore, the response to applied carbachol is reduced by the toxin, indicating that the block of synaptic transmission is due to a decreased response of the postsynaptic membrane. Peroxidase-labeled alpha-bungarotoxin is localized to small (0.2- to 0.5-micrometers diameter) patches beneath synaptic boutons. Peroxidase reaction product is restricted to regions of the synaptic cleft just opposite the active zones of the presynaptic terminal. In addition, peroxidase-labeled antibodies against Torpedo acetylcholine receptor bind exclusively to these same synaptic regions; evidently these patches are the areas at which nicotinic receptors are concentrated at synaptic contacts on sympathetic neurons.