Besides its function at cholinergic synapses, acetylcholinesterase (AChE) exerts structural functions on neural differentiation, independent of its enzymatic activity. To elucidate such functions, we have previously heterologously expressed AChE in histotypic retinal reaggregates, revealing strong effects on their histogenesis, particularly on Müller glia processes. To further resolve these findings at a less complex cellular level, in this study we transfected adherent retinal cells of the chick embryo after 2 days i.c. with a sense pSVK3-AChErab-cDNA expression vector encoding for the entire rabbit AChE gene by calcium phosphate precipitation. Northern blots using digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabbit AChE mRNA in AChE-transfected cells. Western blot analysis established an increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regulation of overall AChE activity. As a corollary, 5'-bromo-2'-deoxyuridine (BrdU) studies showed a decrease in cell proliferation. Exploring changes of the Müller glia, the cytoskeletal protein vimentin was found to be increased in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE in chicken retinal monolayers exerts a structural function on glial cytoskeletal organization, independent of AChE activity.