The cryptic stereochemistry of one-carbon transformations in berberine biosynthesis has been elucidated by the chiral methyl group methodology and tritium NMR spectroscopy. The transfer of the methyl group of AdoMet to oxygen and nitrogen occurs with complete inversion of configuration. The oxidative formation of the berberine bridge from the N-methyl group of reticuline involves removal of a methyl hydrogen with a primary kinetic isotope effect k(H)/k(D) = 4.0 and its replacement by the phenyl group in an inversion mode. The subsequent aromatization catalyzed by (S)-tetrahydroprotoberberine oxidase (STOX) involves nonstereospecific hydrogen removal from C-8 with little or no isotope effect. In the formation of the methylenedioxy bridge, a hydrogen is removed from the methoxy group with k(H)/k(D) > 5 and replaced by the adjacent phenolic oxygen with apparent retention, accompanied by substantial racemization. The subsequent reductive opening of the methylenedioxy bridge of berberine to the methoxy group of jatrorrhizine proceeds stereospecifically, apparently in an inversion mode. Mechanistic interpretations of these findings are discussed.