Deuterated rhamnolipids were produced using strain AD7 of Pseudomonas aeruginosa, which was progressively adapted to increasing levels of deuterium in D2O and carbon substrates. Fourteen different deuterated rhamnolipid structures, including structural isomers, were produced which is similar to normal protonated structures. There were two main products monorhamnolipid Rha-C-10-C-10 and dirhamnolipid Rha(2)-C-10-C-10. The levels of deuteration varied from 16% with 25% D2O + h-glycerol to 90% with 100% D2O + d-glycerol. When d-tetradecane was used with H2O, virtually all the deuterium appeared in the lipid chains while using h-tetradecane + D2O led to the majority of deuterium in the sugars. The adaptation to growth in deuterium appeared to be metabolic since no genetic changes could be found in the key rhamnolipid biosynthetic genes, the rhamnosyl transferases RhlB and RhlC. Deuterated sophorolipids were similarly produced using Candida bombicola and Candida apicola although in this case, no adaptation process was necessary. Up to 40 different sophorolipids were produced by these yeasts. However, unlike the rhamnolipids, use of D2O did not lead to any deuteration of the lipid chains, but direct incorporation into the lipid was achieved using d-isostearic acid. The results from these experiments show the feasibility of producing deuterated bioactive compounds from microorganisms coupled with the possibility of manipulating the pattern of labelling through judicious use of different deuterated substrates.