Here, we introduce the concept of the "seleno effect" in the study of oxidoreductases that catalyze thiol/disulfide exchange reactions. In these reactions, selenium can replace sulfur as a nucleophile, electrophile, or leaving group, and the resulting change in rate (the seleno effect) is defined as kS/ kSe. In solution, selenium accelerates the rate of thiol/disulfide exchange regardless of its chemical role (e.g., nucleophile or electrophile). Here we show that this is not the case for enzyme catalyzed reactions and that the magnitude of the seleno effect can differentiate the role of each sulfur atom of a disulfide bond between that of an electrophile or leaving group. We used selenium for sulfur substitution to study the thiol/disulfide exchange step that occurs between the N-terminal redox center and the C-terminal disulfide-containing β-hairpin motif of Plasmodium falciparum thioredoxin reductase (PfTrxR), which has the sequence Gly-Cys535-Gly-Gly-Gly-Lys-Cys540-Gly. We assayed a truncated PfTrxR enzyme missing this C-terminal tail for disulfide-reductase activity using synthetic peptide substrates in which either Cys535 or Cys540 was replaced with selenocysteine (Sec). The results show that substitution of Cys535 with Sec resulted in a nearly 9-fold decrease in the rate of reduction, while substitution of Cys540 resulted in a 1.5-fold increase in the rate of reduction. We also produced full-length, semisynthetic enzymes in which Sec replaced either of these two Cys residues and observed similar results using E. coli thioredoxin as the substrate. In this assay, the observed seleno effect ( kS/ kSe) for the C535U mutant was 7.4, and that for the C540U mutant was 0.2.