The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC50 = 48 nM) in stimulating 86Rb+ influx into M10 cells, which express the nicotinic receptor subtype comprising alpha 4 and beta 2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC50 = 140 nM). alpha-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC50 of 3.9 microM, whereas alpha 7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 microM for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (-)-nicotine and approximately 20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.