Circular dichroism and proton nuclear magnetic resonance measurements were made to clarify how reduction and reduction plus alkylation of the intrachain disulfide bond affect the conformation and stability of the constant (CL) fragment of the immunoglobulin light chain. The pH titration behavior of the two histidine residues in the intact CL, reduced CL, and reduced and alkylated CL fragments were followed by proton nuclear magnetic resonance spectroscopy. It was shown that reduction of the intrachain disulfide bond does not affect the solution conformation of the immunoglobulin fold, whereas reduction and alkylation results in extensive unfolding of the protein molecule. These results are consistent with the previous results obtained by Goto and Hamaguchi [1979) J. Biochem. 86, 1433-1441) using circular dichroism, fluorescence, and titration of SH groups. The pH-induced unfolding of the reduced CL fragment was compared with that of the intact CL fragment. The stability of the intact CL fragment to acid was much greater than that of the reduced CL fragment. The equilibria of the unfolding by acid could be explained by assuming that the same ionizable groups participate in the unfolding for the reduced CL fragment as for the intact CL fragment, and that the stability of the intact CL fragment is 100 times greater than that of the reduced CL fragment. In the alkaline pH region, the reduced CL fragment was unfolded above pH 8.5, while the intact CL fragment was not unfolded until pH 11.6. The kinetics of the unfolding by alkali of the reduced CL fragment was also studied. The unfolding by alkali of the reduced CL fragment could be explained by assuming that the reduced CL molecule can no longer adopt the folded conformation when either or both SH groups ionize.