Using autoradiography two types of 3[H]epibatidine binding in rat brain was found, the by far most widespread was nicotine displaceable, and represented nicotinic cholinergic receptors, probably the two subtypes alpha4beta2 and alpha3beta4. Using acetylcholine and nicotine in concentrations able to displace 3[H]epibatidine from the alpha4beta2 and alpha3beta4 subunits, in a few brain structures a non-acetylcholine and non-nicotine displaceable 3[H]epibatidine binding was found. The binding was almost exclusively localised to the medial and lateral habenula, interpeduncular nucleus and pineal gland; at 0.1 nM 3[H]epibatidine it represented about 40% of the total 3[H]epibatidine binding. A literature search directed at neurotransmitters in habenula did not demonstrate any neurotransmitter with this anatomical distribution in the brain, suggesting that the non-acetylcholine displaceable 3[H]epibatidine binding represents a binding site, not previously described.