The pathway for the transfer of NADH from one dehydrogenase (E1) to another dehydrogenase (E2) has been investigated by studying the E2-catalyzed reduction of S2 by NADH. The experimental conditions are that the concentration of E1 exceeds that of NADH, which in turn is very much greater than E2; hence, the concentration of free (aqueous) NADH is exceedingly low. The rate of reduction of S2 will hence be very slow if unliganded aqueous NADH is required for the E2-catalyzed reaction. Our results with eight dehydrogenases are entirely consistent with the direct transfer of NADH between E1 and E2 whenever the two enzymes transfer hydrogen via opposite faces (A and B) of the nicotinamide ring. Whenever the two enzymes are both A or both B, NADH transfer occurs only via the aqueous solvent. Some mechanistic inferences and their possible physiological significance are discussed.