The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system that is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered have comparable properties, but functionally separated through their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, while TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with DTNB, lipoamide and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino acid truncated TrxR2 was almost as efficient as full-length TrxR2 in reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2, but not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor for TrxR1 while another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificities and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.