Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. ␣ Subunits, together with 2 and/or 4 subunits, form ligand-binding sites at ␣/ subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the ␣-conotoxin signal sequence and 3 -untranslated sequence region, we have cloned a novel gene from the fisheating snail, Conus bullatus; the gene codes for a previously unreported ␣-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, ␣-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for ␣3-and chimeric ␣6-containing nAChRs; BuIA blocks ␣6/␣32 nAChRs with a 40,000-fold lower IC 50 than ␣42 nAChRs. The kinetics of toxin unblock are dependent on the  subunit. nAChRs with a 4 subunit have very slow off-times, compared with the corresponding 2 subunit-containing nAChR. In each instance, rat ␣x4 may be distinguished from rat ␣x2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse ␣32 to mouse ␣34, and human ␣32 to human ␣34, indicating that the  subunit dependence extends across species. Thus, ␣-conotoxin BuIA also represents a novel probe for distinguishing between 2-and 4-containing nAChRs.