alpha-Conotoxin MII (CtxMII), a peptide toxin from the venom of the predatory cone snail Conus magus, displays an unusual nicotinic pharmacology. Specific binding of a radioiodinated derivative ((125)I-alpha-CtxMII) was identified in brain region homogenates and tissue sections. Quantitative autoradiography indicated that (125)I-alpha-CtxMII binding sites have an unique pharmacological profile and distribution in mouse brain, being largely confined to the superficial layers of the superior colliculus, nigrostriatal pathway, optic tract, olivary pretectal, and mediolateral and dorsolateral geniculate nuclei. Expression of alpha-CtxMII binding sites in the nigrostriatal pathway, combined with evidence for alpha-CtxMII-sensitivity of nicotine-induced [(3)H]dopamine release in rodent striatal preparations indicates that (125)I-alpha-CtxMII binding nicotinic acetylcholine receptors are likely to be physiologically important. Unlabeled alpha-CtxMII potently (K(i) 10 microM) interacted with (125)I-alpha-bungarotoxin and (-)-[(3)H]nicotine binding sites, confirming this compound's novel nicotinic pharmacology. Quantitative autoradiography revealed that alpha-CtxMII binds with high affinity at a subset of [(3)H]epibatidine binding sites with relatively low cytisine affinity ("cytisine-resistant" sites), resolving [(3)H]epibatidine binding into three different populations, each probably corresponding to a receptor subtype. The majority population seems to correspond to that which binds nicotine and cytisine with high affinity ("cytisine-sensitive" sites). Comparison of the cytisine-resistant population's distribution with that of alpha3 subunit mRNA expression suggests that the fractions both more and less sensitive to alpha-CtxMII probably contain the alpha3 subunit, perhaps in combination with different beta subunits.