To elucidate the role of leucine 134 of Bacillus licheniformis nucleotide exchange factor (BlGrpE), site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK), whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the α-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.