Esterase D-1 (carboxylesterase; carboxylic-ester hydrolase, EC 3.1.1.1) was purified to homogeneity and esterase D-2 was highly purified from human erythrocytes. A new procedure, which included fractionation with ammonium sulfate, hydrophobic chromatography on a Toyopearl HW-65 column, and chromatographies on CM-cellulose and hydroxylapatite columns, was developed. Esterases D-1 and D-2 were purified about 9000- and 5600-fold over the precipitates with 65% saturated ammonium sulfate in 14 and 35% yields, respectively. The minimum molecular weights of esterases D-1 and D-2 were estimated to be 35,000 based on the mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without 2-mercaptoethanol. The molecular weights of both enzymes were calculated to be 76,000 by gel filtration. These findings indicated that these two enzymes consisted of dimer without an intermolecular disulfide bond(s). Amino acid analysis of esterase D-1 showed that the total residues of aspartic acid plus asparagine, glutamic acid plus glutamine, glycine, and leucine represent about 40% of the total amino acid residues. Esterases D-1 and D-2 have almost identical biochemical characteristics, including Km values, sensitivities to sulfhydryl reagents, and molecular weights. Esterase D-2 cross-reacted with a rabbit antibody raised against the purified esterase D-1.