Recombinant acetylcholinesterases (AChE) are produced at systematically different levels, depending on the enzyme species. To identify the cause of this difference, we designed expression vectors that differed only by the central region of the coding sequence, encoding Torpedo, rat, and Bungarus AChEs and two reciprocal rat/Bungarus and Bungarus/rat chimeras. We found that folding is a limiting factor in the case of Torpedo AChE and the chimeras, for which only a limited fraction of the synthesized polypeptides becomes active and is secreted. In contrast, the fact that rat AChE is less well produced than Bungarus AChE reflects the levels of their respective mRNAs, which seem to be controlled by their transcription rates. A similar difference was observed in the coding and noncoding orientations; it seems to depend on multiple cis-elements. Using CAT constructs, we found that a DNA fragment from the Bungarus AChE gene stimulates expression of the reporter protein, whereas a homologous fragment from the rat AChE gene had no influence. This stimulating effect appears different from that of classical enhancers, although its mechanism remains unknown. In any case, the present results demonstrate that the coding region contributes to control the level of gene expression.