Reduction of the antioxidant lipoic acid has been proposed to be catalyzed in vivo by lipoamide dehydrogenase (LipDH) or glutathione reductase (GR). We have found that thioredoxin reductase (TR) from calf thymus, calf liver, human placenta, and rat liver efficiently reduced both lipoic acid and lipoamide with Michaelis-Menten type kinetics in NADPH-dependent reactions. In contrast to LipDH, lipoic acid was reduced almost as efficiently as lipoamide. Under equivalent conditions at 20 degrees C, pH 8.0, mammalian TR reduced lipoic acid by NADPH 15 times more efficiently than the corresponding NADH dependent reduction catalyzed by LipDH (297 min-1 for TR vs. 20.3 min-1 for LipDH). Moreover, TR was 2.5 times faster in reducing lipoic acid with NADPH than in catalyzing the reverse reaction (oxidation of dihydrolipoic acid with NADP+). In contrast, LipDH was only 0.048 times as efficient in the forward reaction as compared to the reverse reaction (using NADH and NAD+). We conclude that all or part of the previously described NADPH-dependent lipoamide dehydrogenase (diaphorase) activities in mammalian systems should be attributed to TR. Our results suggest that in mammalian cells a significant part of the therapeutically important reduction of lipoic acid is catalyzed by thioredoxin reductase.