Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α-amylase/subtilisin inhibitor (BASI), two loops in barley thioredoxin h2 (HvTrxh2), containing an invariant cis-proline (⁸⁶EAMP⁸⁹) and a conserved glycine (¹⁰⁴VGA¹⁰⁶), surround the active site cysteines ( ⁴⁵WCGPC⁴⁹) and contribute to binding of BASI through backbone-backbone hydrogen bonds [Maeda, K., Hägglund, P., Finnie, C., Svensson, B., and Henriksen, A. (2006) Structure 14, 1701-1710]. This study involves mutational analysis of key amino acid residues from these two loops in reactions with three protein disulfide substrates, BASI, barley glutathione peroxidase, and bovine insulin as well as with NADPH-dependent barley thioredoxin reductase. HvTrxh2 M88G and M88A adjacent to the invariant cis-proline lost efficiency in both BASI disulfide reduction and recycling by thioredoxin reductase. These effects were further pronounced in M88P lacking a backbone NH group. Remarkably, HvTrxh2 E86R in the same loop displayed overall retained catalytic properties, with the exception of a 3-fold increased activity toward BASI. From the ¹⁰⁴VGA¹⁰⁶ loop, a backbone hydrogen bond donated by A106 appears to be important for target disulfide recognition as A106P lost 90% activity toward BASI but was efficiently recycled by thioredoxin reductase. The findings support important roles in target recognition of backbone-backbone hydrogen bond and electrostatic interactions and are discussed in relation to earlier structural and functional studies of thioredoxins and related proteins.