The riboflavin-producing fungus Eremothecium ashbyii was cultured in various growth media containing high concentrations of deuteriuj, and the product was isolated. The structures of highly deuterated riboflavin, in which at least 13 of 15 nonexchangeable hydrogens were replaced by deuterium, and fully deuterated riboflavin, in which all 15 nonexchangeable sites contained deuterium, were established by NMR and mass spectrometry. The aromatic protons (C-5 and C-8) wer partially substituted in the highly deuterated molecule. Information regarding three areas of the biosynthetic pathway within the microorganism was obtained as a result of the formation of these compounds. Extensive solvent interaction, possibly due to passage of sugar through the transaldolase-transketolase pathway, occurs during formation of the ribityl chain. Limited solvent participation takes place during formation of 6,7-dimethyl-8-ribityllumazine, the immediate precursor of riboflavin. Deuteration of the riboflavin C-6 and C-7 methyl groups indicates significant solvent exchange during the final step of the biosynthetic process.