AbstractAcetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH‐SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non‐neuronal (LLC‐MK2, HeLa, THP‐1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE‐specific activity were higher in neuronal cells than in the non‐neuronal cell types. CGN expressed significantly more AChE activity than SH‐SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH‐SY5Y, CGN, and nerve‐growth factor‐differentiated PC‐12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC‐MK2, fibroblasts, and undifferentiated PC‐12 cells was significantly different than in neuronal cells—AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. © 2005 Wiley‐Liss, Inc.