The hydrogen transfer mechanism of cofactor reduction and recycling processes in the yeast reduction of alpha,beta-unsaturated ketone was studied by using quantitative isotope tracing close to natural abundance measured by (2)H NMR. In the reaction, the active cofactor is NADPH. The cofactor-transferred hydride attacks the beta sp(2) carbon of the enone carbonyl while water hydrogen is transferred to the alpha position. The reductant involved in the reaction depends on the quantity of yeast. When the amount of yeast is very large, the enzymes use preferentially certain unidentified substance stored in the yeast cells rather than the added glucose as electron donor. In this case, the hydrogen transferred by the cofactor is mainly of water origin. When the yeast amount is low, the added glucose is more efficiently used by the enzymes as electron donor and its hydrogen atoms bound to C-1 and C-3 are delivered to the substrate.