A soluble NAD-dependent hydrogenase contained in Alcaligenes eutrophus was evaluated as a coenzyme regenerating catalyst in an organic-aqueous two-phase (predominantly organic) system. The horse-liver alcohol-dehydrogenase (HLADH) catalyzed reduction of cyclohexanone to cyclohexanol was used as a model reaction. The impact of different solvents (selected to span a large variety of principal properties) on the stability and activity of the HLADH, using substrate-driven regeneration, was studied. Solvents suitable for the HLADH were then selected for an evaluation of the hydrogenase-driven coenzyme regeneration. Hydrophobic solvents such as heptane, toluene, and 1,1,1-trichloroethane were found to be suitable for the coupled reactions catalyzed by HLADH and hydrogenase. Nonimmobilized cells, permeabilized with cetyl-trimethyl-ammonium bromide, were the most efficient preparation for the regeneration of NADH. The use of this preparation in heptane (10% water) was optimized with respect to the yield obtained in the HLADH-catalyzed reduction of cyclohexanone. Using the optimized conditions, yields of 99% cyclohexanol were obtained.