The nuclear magnetic resonance study of site-specific natural isotope fractionation (SNIF-NMR) produced in the glycolytic conversion of glucose into ethanol and glycerol provides isotopic transfer coefficients, a ij , which relate sites i in the products to sites j in the reactants. The isotopic connection between the carbon-bound hydrogens of glycerol and those of glucose and water in fermentation reactions carried out with Saccharomyces cerevisiae has been investigated. The a ij coefficients provide mechanistic information on the genealogy of the glycerol hydrogens, on the relative rates of triose phosphate isomerization and reduction of dihydroxyacetone phosphate into glycerol 3-phosphate, on the stereospecificity of the reduction, on the percentages of intra-and intermolecular hydrogen transfer occurring in the course of the reaction, and on the order of magnitude of the active overall kinetic and thermodynamic isotope effects. Thus a close connection is determined between sites 3 pro-R of glycerol (stereospecific numbering) and H 6 pro-R and to a lesser extent H 2 of glucose and between site 3 pro-S of glycerol and H 6 pro-S and H 1 of glucose. Moreover site 2 of glycerol, which exhibits a strong correlation with water is also partly connected with glucose. Possible changes in the values of the isotopic transfer coefficients, as a function of the composition of the medium or of the environmental conditions, enable mechanistic perturbations such as variations in the percentage of intermolecular transfers to be detected. Analyzed in terms of stereospecificity of the reduction step the isotopic results provide a direct chemical shift assignment of the enantiomeric pairs (1 S , 3 R ) and (1 R , 3 S ) of glycerol triacetate. The influence of added bisulfite, which strongly increases the yield in glycerol is estimated. The isotopic characterization of the bioconversion producing both ethanol and glycerol has been extended to the determination of the carbon isotopic parameters by isotope ratio mass spectrometry. Although it usually occurs as a byproduct of the fermentation, glycerol can be considered as a useful complementary probe for characterizing the glycolytic pathway and for inferring various properties of the carbohydrate precursors.