AbstractThree fractions of human liver acylcholine acyl‐hydrolasc (cholinesterase, EC 3.1.1.8.) were separated by chromatography on DEAE cellulose and by gel filtration on Sephadex G‐200. One fraction (II) was identical with human serum cholinesterase with respect to enzymatic properties (substrate and inhibitor specificity, pH dependence, activation by ammonium ions), to solubility in the presence of zinc ions, and to electrophoretic mobility beforc and after removal of sialic acid. Since as much blood as possible was removed from the liver before extraction, it seems unlikely that the fraction originates from plasma. The other fractions (Id and Ie) wcre also identical Jvith serum cholinesterase with respect to enzymatic properties, but differed with respect to molecular properties. They were not sialo‐proteins, and their electrophoretic mobilities differed from that of native and of neuraminidase‐treated serum cholinesterase. Separation on Sephadex G‐200 indicates a lower molecular weight of fraction Ic than of fraction Id and II. The findings are consistent uith the assumption that serum cholinesterase is produced in the liver with fraction Id and Ie as precursors and fraction II as the final product.