Methyllycaconitine (MLA), a natural toxin from Delphinium seeds, was investigated for its ability to antagonize nicotinic responses in several preparations representing different subtypes of neuronal nicotinic acetylcholine receptor. A presynaptic nicotinic receptor mediating dopamine release from rat striatal synaptosomes was blocked by 10 muM MLA, in good agreement with its Ki of 4 muM for inhibition of [(3)H]nicotine binding to striatal membranes. Nicotinic responses in rat superior cervical ganglia were similarly blocked by MLA, and this inhibition was readily reversible. Functional expression of the chick alpha3nalpha1 and alpha4nalpha1 receptor subtypes in Xenopus oocytes confirmed that MLA is a nicotinic antagonist at both receptor subtypes, with IC(50) values of 0.08 and 0.65 muM, respectively. Inhibition by MLA was voltage independent and competitive with agonist concentration. Thus MLA is a useful addition to the nicotinic pharmacopoeia.