In order to characterize the properties of nicotinic acetylcholine receptor (nAChR) subtypes in the CNS, the enantiomers of nicotine, nornicotine and anatoxin-a were studied for their ability to displace (-)-[3H]nicotine binding to membranes and solubilized preparations of different brain regions of rats. In hippocampal membranes, (-)-[3H]nicotine binding was stereoselectively displaced from two sites by (+)- and (-)-nicotine, as well as by (+)- and (-)-anatoxin-a. (-)-Nicotine displayed a larger proportion of high affinity binding sites than did (+)-nicotine, while the proportions of high and low affinity binding sites for (+)-anatoxin-a was the same as that for (-)-anatoxin-a. In cerebellar membranes, the (-)-[3H]nicotine binding was stereoselectively displaced from a single binding site by nicotine and anatoxin-a with Ki values that did not correspond with their KH and KL values observed in hippocampus. The (-)-[3H]-nicotine binding was displaced from a single site by both (+)- and (-)-nornicotine with similar Ki values in both hippocampal and cerebellar membranes. In Triton X-100 solubilized preparations, the (-)-[3H]nicotine binding was displaced from a single site by all of the drugs tested and the Ki values for each individual drug were similar in the cortex, hippocampus and cerebellum. These results provided further evidence for pharmacological heterogeneity of membrane bound nAChRs and clearly indicated that detergent solubilization changed the binding properties of nAChRs in rat brain.