Biosynthesis of the isoprenyl units of the Diels-Alder type adducts in Morus alba callus cultures has been examined by comparing their biosynthesis with that of β-sitosterol (3) produced in the same callus cultures. [1-^<13>C]- and [2-^<13>C]Acetates administered to Morus alba cell cultures were incorporated intact into the isoprenyl unit of 3. [2-^<13>C]Mevalonolactone was also incorporated intact into the expected positions of 3. In the case of the Diels-Alder type adduct chalcomoracin (1), the acetate incorporated into the isoprenyl unit was not the intact acetate administered, but resulted from reorganization from the original acetate through the TCA cycle. Furthermore, [2-^<13>C]mevalonolactone was not incorporated into the isoprenyl unit of 1. These different ways of the incorporation of acetate and mevalonate into the isoprenyl units of 1 and 3 suggest that the mevalonate biosynthesis in 1 and 3 may proceed through a different time schedule. In an analogous experiment with [2-^<13>C]-L-leucine, the incorporation of the ^<13>C-labeled carbon into the isoprenyl units was not observed on either 1 or 3, but the ^<13>C-labeling was incorporated into the polyketide-derived aromatic moieties of 1 as in the case of [1-^<13>C]acetate. This result indicates that the administered L-leucine was metabolized in the cell cultures to [1-^<13>C]acetyl CoA, which then participated in the polyketide synthesis. Reexamination of the biosynthesis of the isoprenyl units of 1 by the pulse administration of [2-^<13>C]acetate revealed that the randomization of the ^<13>C-labeling between cis- and trans-methyls has taken place presumably due to the isomerization of the prenyl group to the diene form for the Diels-Alder type reaction.