To learn more about the evolution of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) 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. On the basis of evidence from kinetics, pharmacology, molecular biology and molecular modeling, we confirm that the enzyme is AChE. Sequence analysis also suggests that the cDNA is AChET and should be able to code for all three globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4); and, assemble into asymmetric forms in association with the collagenic subunit colQ. 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 cells, amphiphilic G1a and non-amphiphilic G4na forms are found. Amphiphilic G2a and non-amphiphilic G4na forms are secreted into the medium. When the catalytic subunit is co-expressed with rat colQ the A12 form of the enzyme is expressed. Collagenase digestion of the A12 AChE at 37°C produces a lytic G4 form; 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 will discuss the relevance of our results to the evolution of the cholinesterases in general, in deuterostome invertebrates, and in chordates including vertebrates.