Esterase from human intestinal mucosa was purified 210 fold by solubilization with Triton X-100, chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite, and isoelectric focusing. The purified esterase showed a single band by polyacrylamide gel electrophoresis. The molecular weight of the purified esterase was estimated to be about 55,000 by gel filtration on Sephadex G-150, and the isoelectric point was 5.02. The purified esterase was strongly inhibited by diethyl p-nitrophenyl phosphate (E-600) and diisopropyl fluorophosphate (DFP), and was not inhibited by eserine sulfate and p-chloromercuribenzoate. The purified esterase from human intestinal mucosa was found to be one of the carboxylesterases. The purified esterase hydrolyzed ester-type drugs, i.e., aspirin, clofibrate, indanyl carbenicillin and procaine, but did not hydrolyze amide-type drugs and choline-type drugs.