In vivo microdialysis was used to investigate nicotinic receptor-mediated acetylcholine release in the hippocampus, frontal cortex, and striatum of freely moving rats. Intraperitoneal administration of (−)-nicotine increased the release of acetylcholine in the hippocampus and frontal cortex but not in the striatum. (−)-Nicotine exhibited a bell-shaped dose–response relationship, and showed attenuation of response at the highest dose (5.0 mg/kg i.p.) in both the hippocampus and frontal cortex. In the hippocampus, (−)-nicotine (1.0 mg/kg i.p.)-induced increase of acetylcholine release was blocked by pretreatment with the centrally acting nicotinic receptor channel blocker, mecamylamine (1.0 mg/kg i.p.), but not by hexamethonium (5.0 mg/kg i.p.), suggesting that the effects of (−)-nicotine were mediated by the central nicotinic receptor. ( S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418, 1.0 and 5.0 mg/kg i.p.), reported to be a selective agonist for α4 β2 nicotinic receptor subunits, also enhanced the release of acetylcholine in the hippocampus, while 3-(2,4-dimethoxybenzlidene)-anabaseine (GTS-21, 1.0 and 5.0 mg/kg i.p.), which has high affinity for the α7 nicotinic receptor subunit, was without effect. The natural alkaloids isolated from plants, (−)-cytisine and (−)-lobeline, had little effect on acetylcholine release from the hippocampus. A competitive antagonist for α4 β2 subunits of the nicotinic receptor, dihydro- β-erythroidine, and a partial agonist for the β2 subunit-containing nicotinic receptor, (−)-cytisine, inhibited (−)-nicotine-induced increase of acetylcholine release from the hippocampus, whereas a selective antagonist for the α7 subunit, methyllycaconitine, and a partial agonist for the α3 subunit-containing nicotinic receptor, (−)-lobeline, did not. These results indicate that there are certain differences among brain regions in the response of nicotinic receptor-mediated acetylcholine release and that (−)-nicotine-induced acetylcholine release in the rat hippocampus may be attributed to activation of the α4 β2 nicotinic receptor subunits.