The oxidative refolding pathway of a three-disulfide mutant of bovine pancreatic ribonuclease A (RNase A) from the fully reduced unfolded form to the native state has been studied by using oxidized and reduced dithiothreitol as the redox reagents at pH 8.0 and 25 degrees C. This mutant was prepared by replacing Cys40 and Cys95 in RNase A with alanines while maintaining the other three native disulfide bonds to mimic one of the two major three-disulfide intermediates (des-[40-95]) observed in the regeneration of wild-type RNase A. The kinetics of refolding of this mutant were measured by quenching the regeneration reaction at various times with a rapid blocking reagent, 2-aminoethyl methanethiosulfonate (AEMTS), fractionating the disulfide intermediates by using cation-exchange HPLC, and analyzing the time course of each group of disulfide species. It was found that the disulfide intermediates formed during regeneration reach a steady-state distribution after a short period of preequilibration similar to that in the regeneration of wild-type RNase A. The experimental data acquired under different redox conditions were fit to a kinetic model with a steady-state treatment. The fitted results indicate that this mutant refolds through a rate-determining step which involves the oxidation of certain two-disulfide species to form a putative three-disulfide species which proceeds rapidly to the native protein. A rough estimation suggests that this pathway could constitute no more than 5% of the major pathway leading to the formation of des-[40-95] (the major three-disulfide intermediate formed) in the regeneration of wild-type RNase A. Several kinetic constants pertaining to the oxidation and reduction of various disulfide intermediates were compared with those obtained in the regeneration studies of wild-type RNase A to gain further understanding about the folding pathways of RNase A. Comparisons are also given for the oxidative refolding studies of several other three disulfide bond proteins, suggesting that the formation of a large number of disulfide-bonded intermediates during oxidative refolding is probably a common feature for most proteins.