The activity of α-conotoxin (α-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic α-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 μM, log IC50 = 0.4 ± 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. α-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, α-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 μM. Bovine chromaffin cells are known to contain the α3β4, α7, and (possibly) α3β4α5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by α-bungarotoxin, indicating that α7 nicotinic receptors are not involved. We propose that α-CTX ImI interacts selectively with the functional (α3β4 or α3β4α5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.