We have developed a NH(3)/H(2)O(2) two-step method for the recovery of insulin monomers from amyloid fibrils by modulating the cleavage and regeneration of disulfide bonds. Insulin fibrils were disaggregated into insulin A- and B-chains in 14 M (w/v) NH(4)OH for 2 h at 60 °C. Insulin monomers, with a MW of ~5,882 Da, were then regenerated by oxidation of sulfhydryls with 30 % (w/v) H(2)O(2) (10 M) for 12 h at 25 °C. No two A-chains or two B-chains of insulin formed during the oxidation process. Because of the inconformity of the optimal reduction and oxidation temperature, the NH(3)/H(2)O(2) two-step method is more practical than the NH(3)/H(2)O(2) coupling method.