We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the alpha5 subunit in the adult rat brain. Among the regions of brain we surveyed, the alpha5 subunit is associated in approximately 37% of the nAChRs in the hippocampus, approximately 24% of the nAChRs in striatum, and 11-16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the alpha5 subunit is associated with alpha4beta2* nAChRs exclusively. Importantly, in contrast to alpha4beta2 nAChRs, which are increased by 37-85% after chronic administration of nicotine, the alpha4beta2alpha5 receptors are not increased by nicotine treatment. These data thus indicate that the alpha4beta2alpha5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo, which suggests an important regulatory role for the alpha5 subunit. To the extent that nicotine-induced up-regulation of alpha4beta2 nAChRs is involved in nicotine addiction, the resistance of the alpha4beta2alpha5 subtype to up-regulation may have important implications for nicotine addiction.