A monoclonal antibody (MAb) raised against human acetylcholinesterase was found to have catalytic activity. A similar phenomenon was observed in a polyclonal antibody raised against the same antigen. The antibodies were demonstrated to be pure, and no contamination with either acetylcholinesterase or butyrylcholinesterase was found. Both antibodies hydrolyzed acetylthiocholine, an acetylcholinesterase substrate, and the MAb followed Michaelis-Menten kinetics. Six other MAbs and one other polyclonal antibody showed no evidence of catalytic activity. Acetylcholinesterase is a key component in the transmission of the nerve impulse, and is also expressed nonsynaptically during embryonic development, and abnormalities in expression are seen in neural tumors and degenerative disorders. This unusual expression is believed to be associated with a novel function of the enzyme related to differentiation and cell adhesion. Autoantibodies to acetylcholinesterase have been observed in a variety of neurologic, muscular, and autoimmune disorders. In an investigation of the possible role of acetylcholinesterase in cell adhesion, we showed that the enzyme promoted neurite outgrowth in neuroblastoma cell lines, and conversely, that certain antiacetylcolinesterase antibodies abrogated cell-substrate adhesion. Interestingly, the antibodies most effective in this regard were catalytic. Preliminary epitope analysis indicated a conformational epitope in the N-terminal domain. This domain contains the active site within a deep gorge and the peripheral anionic site at the rim of the gorge. Peripheral-site inhibitors, but not active-site inhibitors, also interfered with adhesion, and competed with the catalytic monoclonal binding to acetylcholinesterase, indicating that the epitope recognized is associated with the peripheral anionic site. The inhibitor data also support the supposition that catalysis in these antibodies may have arisen from stable complexation of acetylcholinesterase with an inhibitor. We conclude that the catalytic antiacetylcholinesterase antibody interacts with structures associated with the peripheral anionic site, thus defining a novel site on the molecule involved in cell adhesion. This finding has implications for our understanding of the potential importance of this peripheral site in a variety of congenital, neoplastic, and degenerative conditions.