1. The activation and desensitization properties of nicotinic acetylcholine receptor (nAChR) channels were examined in acutely isolated medial habenula (MHb) neurons using whole cell patch-clamp recordings. nAChR- mediated currents were evoked by applying known concentrations of nicotinic agonists using rapid solution exchange techniques. 2. At a membrane potential of -60 mV, nAChR currents were observed above a concentration of ≃100 nM nicotine. The peak current amplitude at low doses of agonist was proportional to the square of the concentration of nicotine, indicating that at least two molecules of agonist were required for channel opening. The concentration of nicotine required for half-maximal nAChR activation was estimated as 77 μM from a complete concentration-response curve. 3. During the continuous activation (2-5 s) of nAChRs by high concentrations of nicotine (300 μM), the current desensitized rapidly and extensively. The desensitization phase was described by the sum of two exponentials, with time constants of 210 and 1,435 ms. The fast component comprised 74% of the desensitizing phase of the current. Recovery from desensitization induced by 2-s applications of 300 μM nicotine was also fast and could be reasonably well described by a single exponential with a time constant of ≃800 ms. Both the time courses of desensitization and recovery from desensitization were slightly slower at positive membrane potentials. 4. Incubation of neurons with low concentrations of nicotine (100 nM-10 μM) caused a slowly developing but pronounced desensitization of the nAChRs. In these cases desensitization was assessed from the reduction in the amplitude of the peak nicotinic current induced by repetitively applied pulses of a higher test concentration of agonist. A 5-min continuous exposure to 1 μM nicotine reduced the amplitude of the acetylcholine (30 μM, 1 s) test response to <30% of its control value. As with higher concentrations of nicotine, the onset of the desensitization induced by 1 μM nicotine was biexponential, with fast and slow time constants of 15 s and 1.74 min, respectively. Recovery from the desensitization induced by these longer applications of nicotine was much slower than that observed with the brief pulses of high concentrations of nicotine. The concentration required for half-maximal desensitization after a 5-min incubation was ≃300 nM. 5. Peak nAChR currents were ≃85% smaller at +40 mV compared with -40 mV. The receptors that do not open at positive potentials desensitize almost as well as they would at negative potentials after channel opening. Thus, under certain conditions, nAChR channels in MHb neurons may desensitize without opening. Desensitization from the open state, however, is likely to be the major transition at physiologically relevant membrane potentials. 6. The large difference between the effective concentrations of nicotine required for channel opening and desensitization has important implications for understanding how the levels of nicotine associated with tobacco inhalation lead to long-term behavioral changes. nAChR channels in MHb neurons will be strongly desensitized by smoking- related concentrations of nicotine. This effect occurs over several minutes and may result in acute tolerance to subsequent doses of nicotine. Long-term disruption of the endogenous activation of nAChRs due to desensitization could underlie the observed upregulation of receptors after chronic nicotine use.