The acetylcholine esterase inhibitor (-)-physostigmine has been shown to act as agonist on nicotinic acetylcholine receptors from muscle and brain, by binding to sites on the alpha-polypeptide that are distinct from those for the natural transmitter acetylcholine (Schröder et al., 1994). In the present report we show that (-)-physostigmine, galanthamine, and the morphine derivative codeine activate single-channel currents in outside-out patches excised from clonal rat pheochromocytoma (PC12) cells. Although several lines of evidence demonstrate that the three alkaloids act on the same channels as acetylcholine, the competitive nicotinic antagonist methyllycaconitine only inhibited channel activation by acetylcholine but not by (-)-physostigmine, galanthamine or codeine. In contrast, the monoclonal antibody FK1, which competitively inhibits (-)-physostigmine binding to nicotinic acetylcholine receptors, did not affect channel activation by acetylcholine but inhibited activation by (-)-physostigmine, galanthamine and codeine. The three alkaloids therefore act via binding sites distinct from those for acetylcholine, in a 'noncompetitive' fashion. The potency of (-)-physostigmine and related compounds to act as a noncompetitive agonist is unrelated to the level of acetylcholine esterase inhibition induced by these drugs. (-)-Physostigmine, galanthamine and codeine do not evoke sizable whole-cell currents, which is due to the combined effects of low open-channel probability, slow onset and slow inactivation of response. In contrast, they sensitize PC12 cell nicotinic receptors in their submaximal response to acetylcholine. While the abundance of nicotinic acetylcholine receptor isoforms expressed in PC12 cells excludes identification of specific nicotinic acetylcholine receptor subtypes that interact with noncompetitive agonists, the identical patterns of single-channel current amplitudes observed with acetylcholine and with noncompetitive agonists suggested that all PC12 cell nicotinic acetylcholine receptor subtypes that respond to acetylcholine also respond to noncompetitive agonist. The action of noncompetitive agonists therefore seems to be highly conserved between nicotinic acetylcholine receptor subtypes, in agreement with the high level of structural conservation in the sequence region harboring major elements of this site.