The enzyme splitting pantethine into pantothenic acid and cystamine has been purified about 3000 times starting from horse kidney. The purification procedure is reported. The enzyme, whose optimal activity lies in the pH range 4.0–5.5, requires the presence of a reduced thiol for the full activity: mercaptoethanol and dithiothreitol give the higher effect. This activation seems not to be specific. The enzyme has a K_m value for the substrate of the order of 5 mM and shows inhibition by excess substrate and by products. Substrate specificity studies show that the enzyme splits only the intact substrate molecule, which is not hydrolyzed by several hydrolytic enzymes, even with a broad specificity (as bacterial pronase and subtilisin). The enzyme could be responsible for the production in vivo of cysteamine, which is known to be oxydized by a specific oxygenase, isolated recently in this laboratory.