The determination of the nuclear magnetic resonance (NMR) solution structured of the mixed disulfide between the mutant Escherichia coli glutaredoxin Grx(C14S) and glutathione (GSH), Grx(C14S)-SG, is described, the binding site for GSH on Grx(C14S) is located, and the non-bonding interactions between -SG and the protein are characterized. Based on nearly complete sequence-specific NMR assignments, 1010 nuclear Overhauser enhancement upper distance constraints and 116 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by energy minimization in a waterbath with the AMBER force field in the program OPAL. The -SG moiety was found to be localized on the surface of the protein in a cleft bounded by the amino acid residues Y13, T58, V59, Y72, T73 and D74. Hydrogen bonds have been identified between -SG and the residues V59 and T73 of Grx(C14S), and the formation of an additional hydrogen bond with Y72 and electrostatic interactions with the side-chains of D74 and K45 are also compatible with the NMR conformational constraints. Comparison of the reduced and oxidized forms of Grx with Grx(C14S)-SG shows that the mixed disulfide more closely resembles the oxidized form of the protein. Functional implications of this observation are discussed. Comparisons are also made with the related proteins bacteriophage T4 glutaredoxin and glutathione S-transferase.