Plant natural products have served as a prominent source of medicines throughout human history and are still used today as clinically approved pharmaceuticals. However, many medicinal plants that produce useful compounds are slow growing or recalcitrant to cultivation, making it difficult to investigate the underlying genetic/enzymatic machinery responsible for biosynthesis. To better understand the metabolism of bioactive natural products in slow-growing medicinal plants, we used D2O labeling and LC-MS-based metabolomics to explore the biosynthesis of medically relevant alkaloids in three plant species. Our results provide evidence for sites of active biosynthesis for these alkaloids and demonstrate that D2O labeling can be used as a general method to determine sites of active secondary metabolism over relatively short-timescales. We anticipate that these results will facilitate discovery of complete metabolic pathways for plant natural products of medicinal importance, especially for approaches that rely upon transcriptomics and knowledge of active metabolism to identify biosynthetic enzymes. (c) 2018 American Institute of Chemical Engineers