The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and L-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 M GdnHCl to 1 M GdnHCl, and 4,4′-dianilino-1,1′binaphthyl-5,5′-disulfonic acid dipotassium salt binding analysis indicated that the addition of L-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether L-arginine was present. Spontaneous folding at the 1 M GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of L-arginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of L-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether L-arginine was present. Controlled coupling of the effects of GSSG and L-arginine led to the complete refolding of scFv in the stepwise dialysis.