Acetylcholinesterase (AChE; EC 3.1.1.7) is expressed in the central nervous system in multiple molecular forms that may subserve multiple functions and may be selectively lost in neurodegenerative illnesses such as Alzheimer's disease. AChE expression has been studied in primary cultures of developing vertebrate nervous system, but investigation has been limited by the lack of a suitable human CNS surrogate cell model system for in vitro studies and the inability of primary brain cultures to provide large numbers of pure neurons. To develop an in vitro model for studies of neuronal AChE expression and function, we utilized a neuronally committed human teratocarcinoma cell line, NTera 2, that can be induced to differentiate to a post-mitotic CNS neuronal phenotype. We found that NTera 2 cells express multiple molecular forms of AChE that are similar to CNS-derived AChE isoforms in velocity sedimentation profile, anion exchange elution profile, and sensitivity to inhibitors. At least two forms of AChE are expressed (G1 and G4), similar to human and rodent brain, and induction of NTera 2 cell differentiation results in an increased G4/G1 ratio, which is characteristic of mature neurons. As in primary CNS neurons, AChE is present in NTera 2 cells in both the cytosolic fraction and in the outer membrane, and is also released in a soluble form. These observations indicate that NTera 2 cells provide a useful human model system for studies of expression of cell-associated and soluble cell-free AChE in developing and mature human neurons and for elucidating the potential role(s) of acetylcholinesterase metabolism in both normal development and neurodegenerative disease states.