The enantiomeric purity of natural a-monodeuterated enantiomers, (R) and (S) ethanol-1-d₁, in the alcohol produced by sugar fermentation with yeast was studied by ²H NMR using their esters derived from optical mandelic acid. The results of isotope tracing experiments show that the transfer pathways of the two enantiotopic hydrogens of the methylene group are different. It was observed that (S)-deuterium comes only from the medium water. The (R)-deuterium transfered by NADH in alcohol dehydrogenase reduction of the acetaldehyde is of complex origin. Some of them originates from carbon bound hydrogen of the sugar, especially from C4) position of glucose and most of them comes from water. Only a small portion of the NADH deuterium is incorporated indirectly from water through enzyme catalysed exchange between the pro-S site of NADH and flavin. When a carbonyl compound (ethyl acetoacetate) was reduced under the same conditions during the alcoholic fermentation, among the NADH-transfered deuterium, only a small portion comes from water while most comes from the unexchangeable positions of the glucose.