We have developed a NH3/H2O2 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) NH4OH for 2 h at 60 A degrees C. Insulin monomers, with a MW of similar to 5,882 Da, were then regenerated by oxidation of sulfhydryls with 30 % (w/v) H2O2 (10 M) for 12 h at 25 A degrees 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 NH3/H2O2 two-step method is more practical than the NH3/H2O2 coupling method.