To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.