Chronic administration of high doses of nicotine results in axonal degeneration in the central core of the fasciculus retroflexus, a fiber tract connecting the habenulae (Hb) to the interpeduncular nucleus (IPN). An important part of this connection is cholinergic and neurons of origin are located in the medial Hb. We have undertaken the present investigation in order to ascertain whether the cholinergic Hb-IPN neurons are the actual target of nicotine toxicity and to begin studying molecular correlates of this action. In the present report, we demonstrate that 7-day-long continuous administration of nicotine through osmotic minipumps, results in a significant (-13%) decrease in the volume of the medial Hb, where cholinergic neurons projecting to the IPN are located, and in a drop of a specific marker for cholinergic neurons, choline acetyltransferase (ChAT), in Hb (-36%) and IPN (-28%). At various intervals (2-6 days) during continuous nicotine administration, some apoptotic neurons were visualized in the medial Hb by the TUNEL technique. The chronic nicotine treatment also resulted, after 2 days of continuous administration in significant activation of the transcription factor CREB and the ERK/MAPK survival kinase in the Hb, suggesting that these alterations in expression are in some way related to the neurodegenerative/neuroreparative process. The present observations demonstrate that the cholinergic Hb-IPN neurons are a target for nicotine neurotoxicity and confirm the usefulness of the experimental model used here not only to study the consequences of chronic stimulant abuse, but also to study the neurochemistry of the affected neural systems and the role of signaling factors in neurodegenerative and repair mechanisms. Medical relevance of the data on unique vulnerability of the Hb-IPN connection to nicotine in relation to heavy smoking habits, is briefly discussed.