To determine the biosynthesis pathway of the atisine-type diterpenoid alkaloids spiramines A/B and C/D, feeding experiments in in vitro cultured plantlets and enzymatic transformations in cell-free extracts were performed in combination with LCMS and tandem MS analyses. L-[2-(13)C,(15)N]Serine was used in the feeding experiments and enzymatic transformations, and the diterpene spiraminol was identified as a biosynthetic precursor of spiramine alkaloids. The LCMS and tandem MS spectra of the extracts from these experiments indicated that L-[2-(13)C,(15)N]serine was incorporated into spiramines A/B and C/D. The labeled reaction products show that l-serine is the one possible nitrogen source involved in the biosynthesis of atisine-type DAs.