Phenylalanine-1-¹⁴C, -2-¹⁴C and -3-¹⁴C, and malonic acid-2-¹⁴C were efficiently incorporated into mangiferin (1) in Anemarrhena asphodeloides. In the case of feeding with phenylalanine-1-¹⁴C, -2-¹⁴C and malonic acid-2-¹⁴C, the radioactivity of 1 was localized in the phloroglucinol ring. Furthermore, cinnamic acid-3-¹⁴C and p-coumaric acid-2-¹⁴C were also incorporated into 1 and isomangiferin (2), but benzoic acid-, p-hydroxybenzoic acid-and protocatechuic acid-(carboxyl-¹⁴C) were essentially not incorporated into 1 or 2. In addition to the above data, doubly labelled p-coumaric acid was incorporated into 1 without change of the T/¹⁴C ratio. These results show that the aglycone of 1 and 2 was biosynthesized from p-coumarate (C₆-C₃) and two malonates (C₄).