Summary: C′1-esterase was isolated by column chromatography on DEAE and TEAE cellulose of a euglobulin fraction of human serum. The final product, purified 2400-fold with respect to serum, contained 3800 enzyme units/mg of nitrogen, as measured by zero-order hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATE). The purified preparation migrated as an α2-globulin on paper electrophoresis but was heterogeneous in the analytical ultracentrifuge. The susceptibility to hydrolysis of synthetic substrates, listed in decreasing order of activity, was: N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-tyrosine methyl ester, benzoyl-L-arginine ethyl ester, p-toluenesulfonyl-L-arginine methyl ester, and N-acetyl-L-phenylalanine ethyl ester. Nonsusceptible substrates included L-lysine methyl ester. Maximal hydrolysis of ATE occurred at a final concentration of substrate of 5 × 10-2 M (Km = 1.9 × 10-2 M), pH 6.7 to 8.0, ionic strength below 0.20, and 38.8°C (apparent energy of activation = 10,400 calories/mole). No dependency of esterolytic activity on divalent cations was demonstrable. These properties are similar to those of previous preparations of human and guinea pig C′1-esterase obtained either from less purified fractions of serum or from elution of antigen-antibody-complement complexes. Possible explanations for the observed physicochemical heterogeneity are discussed. The function of this highly purified preparation of human C′1-esterase in the complement system is presented in the following article.