Externally added ascorbate or NADH effectively reduced ferricyanide and promoted the exit of Fe3+ originated from acid-destabilized transferrin contained inside endocytic vesicles. The effect of ascorbate was mediated by an ascorbate uptake system, and the effect of NADH was mediated by the membrane-associated oxidoreductase. At physiological concentrations of both ascorbate and NADH, the ascorbate transport and the NADH-oxidoreductase system were additive as measured by the rate of reduction of ferricyanide and by the mobilization of transferrin-associated iron. The results indicate that Fe3+ reduction may occur by a non-enzymatic reaction with ascorbate transported into the vesicle lumen. The ascorbate-mediated reduction of iron derived from transferrin occurring in the endosome could play a major role in cellular iron uptake.