Procedures for the purification of horse and shark liver carbostyl.esterases are reported together with a modified procedure for the purification of sheep liver carboxylesterase. In the procedure for the horse enzyme, the starting material is an acetone powder, whereas for the other two enzymes, it is a chloroform-acetone powder. Each procedure involves ammonium sulphate fractionation of the powder extract, ion-exchange chromatography and gel filtration. The yields of sheep, horse and shark liver carboxylesterases are 200, 230, and 50 mg, respectively.The shark enzyme appears homogeneous on polyacrylamide gel electrophoresis, and in the case of the sheep and horse enzyme preparations the major proportion of the esterase activity occurs in one band, with minor, faster-migrating components,In contrast to the other liver carboxylesterases, the enzyme from shark liver catalyzes the hydrolysis of ethyl butyrate much less efficiently than that of phenyl butyrate.'Shark liver carboxylesterase has one active site per 83,000 daltons, as shown by titration with p-nitrophenyl diethyl phosphate. Sedimentation equilibrium experiments gave a z-average molecular weight of 80,500 daltons, and a second virial coefficient of 8.2 x 10-5 mole cm3 g-2 . There is no evidence of association in the concentration range of ~1-7 g/1. , a result which is in contrast to that found for the liver carboxylesterases from sheep, horse, ox and pig, but is similar to that for the chicken enzyme.With a molecular weight of ~80,000 daltons, shark liver carboxylesterase is larger than the corresponding enzymes from chicken, sheep, horse, ox and pig livers, all of which have a molecular or subunit weight of ~65,000 daltons.The amino acid compositions of the six liver carboxylesterases mentioned above are reported. As would be expected for this homologous series, the compositions show a general similarity. However, there are some significant differences, but the degree to which particular pairs of enzymes differ is consistent with the evolutionary history of the species from which they were isolated.A computer program is described for the calculation of the complete amino acid composition of a protein from the analytical data. The program also derives a molecular weight on the basis of the amino acid composition. The molecular weights of the liver carhoxylesterases of chicken, horse, ox and sheep were found, by this method, to be ~67,000 daltons.