ChAT (choline acetyltransferase) is found in cholinergic neurons where its function is to catalyze the reaction between acetyl-CoA and choline to form the neurotransmitter ACh. The density of cholinergic neurons and levels of ChAT gene expression have been studied extensively in Alzheimer’s Disease (AD). ChAT is encoded by one gene, however through the use of differential splicing and utilization of alternative promoters, multiple transcripts of the protein exist. My studies focus on the proteins transcribed from the M-type mRNA, which yield the common 69-kDa ChAT protein, as well as a primate specific 82-kDa ChAT protein. The two proteins differ by a 118 amino acids amino-terminal extension. Studies previously conducted in this laboratory found that the 82-kDa ChAT contained a nuclear localization signal (NLS) that targeted the protein into the nucleus of cells. Analysis of the primary amino acid sequence of ChAT indicates additional putative nuclear transport domains. In addition to the unknown functional significance of nuclear ChAT, the endogenous expression of 82-kDa ChAT has yet to be shown. Our results highlight novel functional transport domains within the human 69-kDa and 82-kDa ChAT enzymes by employing confocal microscopy. Furthermore, using immunohistochemical techniques, we demonstrate for the first time, the endogenous nuclear expression of 82-kDa ChAT in human neural tissue of neurologically normal and Clinical Dementia Rated (CDR) cases. Moreover, combining functional genomics technology with cell biology, we illustrate that in addition to synthesizing the neurotransmitter ACh, ChAT is involved in the negative regulation of toxic Aβ peptide production. Taken together, these studies further our knowledge of the ChAT isoforms, their cellular localization, their endogenous expression, as well as their direct influence on Aβ peptide production. This research can have implications in diseases such as AD, where the toxic effects of Aβ peptides target cholinergic neurons. These studies were designed to broaden our understanding of the cellular mechanisms governing ChAT isoforms in the presynaptic cholinergic environment. The information collected enhances our understanding of ACh biosynthesis as well as the regulation, modulation, and cell signaling events involving ChAT in the physiological state. Keywords. choline acetyltransferase, cholinergic, nuclear localization signal, nuclear export signal, subcellular distribution, nucleocytoplasmic shuttling protein, nuclear transport, Alzheimer disease, mild-cognitive impairment, immunohistochemistry, amyloid processing, secretases, microarray, Amyloid Processing Protein.