Regional-specific differences in nicotinic acetylcholine receptors (nAChRs) were examined using the whole-cell patch clamp technique in rat medial habenula (MHb) slices. The majority of cells in the ventral two thirds of the MHb responded robustly to local pressure application of nAChR agonists. Mean agonist potency profiles in the middle and ventral thirds of the MHb were similar: cytisine was the most potent agonist and DMPP the weakest, consistent with a significant contribution of the β4 subunit to functional nAChRs in all areas of the MHb. In acutely isolated MHb neurons, the α3β4-selective toxin α-CTx-AuIB (1 μM) reversibly blocked~75% of the nicotine-induced currents, as expected for cells solely expressing α3β4 nAChRs. However, the α3β2-selective toxin, α-CTx-MII (100 nM), blocked a variable fraction (0-90%) of the MHb nicotinic response implying that β2 subunits may contribute to some functional receptors. We suggest that the effects of α-CTx-MII may arise from interaction with α3β2β4 subunit-containing nAChRs. This idea is supported by the findings (1) that α-CTx-MII antagonizes receptors comprised of α3, β2 and β4 subunits in Xenopus oocytes, and (2) that a mutant α-CTx-MII toxin[H12A], which blocks α3β2β4 receptors but not α3β2 or α3β4 nAChRs, also reduces nicotinic currents in some MHb neurons. Overall these data imply that most functional nAChRs on MHb cells contain at least α3 and β4 subunits, and that a variable subpopulation additionally contains the β2 subunit. Copyright (C) 1999 Elsevier Science Ltd.