A comparative study of bile acid components from four lamprey species revealed that mature male chestnut lamprey Ichthyomyzon castaneus can produce 3 keto petromyzonol sulfate (3kPZS) while mature males of Pacific lamprey Lampetra tridentata, river lamprey Lampetra fluviatilis and American brook lamprey Lethenteron appendix produce petromyzonol sulfate (PZS). Identification of 3kPZS from a group of ancient lamprey species and of PZS from recently derived species led to a speculation that differentiation of bile acid biosynthetic systems has taken place during the course of evolution. Further studies on the biological functions of different bile acids in the adult lampreys are required to understand the evolution of chemical communication in lampreys.