It is well established that prostaglandin catabolism involves sequential actions of a 15-hydroxyprostaglandin dehydrogenase, a 15-keto-prostaglandin delta 13-reductase and a 15-ketoprostaglandin reductase. This pathway must be confirmed in never investigated tissues before any enzyme assay is carried out. We have developed a new, simple, rapid and reliable method to investigate catabolizing sequence of prostaglandins based on the tritium kinetic isotope effect which occurs during the oxidation of the 15-hydroxyl group of the prostaglandin into a 15-keto group.