The substrate specificty of pantethinase, the mammalian enzyme which has been isolated from horse kidney cortex, has been studied. The real substrate has been found to be pantetheine in the reduced form, and the involvement of one or more—SH groups of the enzyme is also considered.A number of other compounds, chemically related to pantetheine, has also been tested as substrates. Pantethinase is able to metabolise pantetheine‐4′‐phosphate, with an activity about 10% of that exhibited for pantetheine; it is inactive on the other substances tested (pantothenoyl‐cysteine‐4′‐phosphate, pantothenoylcysteine, pantetheine thiazoline, CoA).The metabolic role of pantethinase seems to be, in the light of the above results, connected with a degradative pathway of pantetheine‐4′‐phosphate, which is known to be one of the intermediates in the biosynthesis of CoA.