Cholinergic neurons express choline acetyltransferase (ChAT) which synthesizes acetylcholine. We show here for the first time that primate-specific 82-kDa ChAT is expressed in nuclei of cholinergic neurons in human brain and spinal cord; isoform-specific antibodies were used to compare localization patterns and temporal expression of the more abundant 69-kDa ChAT and primate-specific 82-kDa ChAT in necropsy tissues. The 82-kDa ChAT co-localizes with 69-kDa ChAT in well-characterized cholinergic areas, but is also found in the claustrum which does not contain 69-kDa ChAT. Cholinergic neuron function changes with increasing age and are targeted in neurodegenerative diseases such as AD, thus we compared expression and subcellular localization of 69- and 82-kDa ChAT in necropsy brain samples from control subjects of varying ages and from Alzheimer disease (AD) subjects. The 82-kDa ChAT protein was expressed in cholinergic neurons in brain from birth until the eighth decade of life and in AD, but the subcellular staining pattern and proportion of neurons that were immunopositive changed with increasing age and in AD.