1. A carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) from human brain extract was prepared to purity using DEAE-cellulose, Sephadex G-200, and fractionation with (NH4)2SO4. The yield was about 20%. 2. Esters of butyric acid were split faster than esters of acetic, propionic and valeric acid, and the enzyme is tentatively designated as a butyrylesterase. Thiocholine esters were split at low rates. 3. The molecular weight was estimated as 340 000 using gel chromatography on Sephadex G-200. In isoelectric focussing the enzyme was resolved into several peaks (pI 4.0--4.7). The low isoelectric point does not seem to be due to terminal sialic acid residues. 4. The enzyme was irreversibly inhibited by diethyl-p-nitrophenyl phosphate (ki = 206 mol-1 - 1 - s-1) and by diisopropylfluorophosphate. The carboxylesterase inhibitor bis-p-nitrophenyl phosphate and eserine did not inhibit the enzyme. 5. The enzyme was progressively inhibited by p-hydroxy-mercuribenzoate, and reactivated by dithiothreitol and 2-mercaptoethanol. N-Ethylmaleimide inactivated the enzyme very slowly, whereas iodoacetate and iodoacetamide were without effect. 6. Ca2+, Mg2+, and Zn2+ or EDTA did not influence the enzyme activity.