The axonal membranes from dog mesenteric nerves were isolated and characterized. They had a high density of [3H]saxitoxin, [3H]rauwolscine and [125I]iodocyanopindolol binding sites, but low densities of [125I] omega-conotoxin (GVIA) and [3H]prazosin binding sites. The microsomal membranes had one of the higher densities of [3H]rauwolscine binding sites in centrifugation fractions. These sites were characterized by saturation, kinetic and competition studies. In competition with oxymetazoline, WB 4101, prazosin, ARC-239, SK&F 104078 and SK&F 104856, these rauwolscine binding sites were alpha 2A-like. Chloroethylclonidine pretreatment did not affect the ability of SK&F 104078 or SK&F 104856 to compete with rauwolscine binding sites on axonal membranes. Bmax values were unchanged by chloroethylclonidine under conditions that also failed to reduce Bmax values of rauwolscine binding to membranes from human platelets. These receptors appear to be alpha 2A adrenoceptors and may represent sites of prejunctional control of neurotransmitter release in the dog. This membrane preparation may be useful in biochemical studies of prejunctional neural receptors.