The activity of alpha-conotoxin (alpha-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 alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.