Peroxiredoxins (Prxs) are a ubiquitous family of thiol peroxidases that protect cells from peroxides and have a putative role in redox signaling. In this study, we investigated the redox properties of human Prx 3, a typical 2-Cys Prx that is localized to the mitochondrial matrix. We found that Prx 3 displayed strong reactivity with H(2)O(2), with a competitive kinetic approach generating a second order rate constant of 2 x 10(7) M(-1) s(-1). This is considerably higher than typical thiols and similar to values for other mammalian 2-Cys Prxs. In contrast, Prx 3 reacted very slowly with the thiol alkylating agents iodoacetamide and N-ethylmaleimide. Using dithiothreitol redox buffers, we measured the redox potential of Prx 3 of -290 mV. This is similar to the redox potential of mitochondrial thioredoxin 2 and is consistent with optimal operation of Prx 3 in the mitochondrial matrix.