A cross-linked ribonuclease A derivative, Lys7-dinitrophenylene-Lys41-ribonuclease A, has been prepared and characterized for ultimate use in protein-folding experiments. Immunochemical assays and spectroscopic measurements demonstrated that the introduction of this artificial cross-link does not perturb the native conformation of ribonuclease A. The cross-linked protein exhibited a reversible thermal transition with Tm = 53 degrees C at pH 2.0, which is 25 degrees C higher than that of unmodified ribonuclease A under the same conditions. The denatured form of the cross-linked ribonuclease A has a conformational chemical potential that is 4.9 kcal/mol higher than that of the denatured form of unmodified ribonuclease A at 40 degrees C and pH 2.0, assuming that the cross-linked and the unmodified proteins have the same conformational chemical potential in the native conformation. This is in good agreement with a theoretical value of 5.2 kcal/mol, calculated from the reduction of chain entropy of the denatured form upon introduction of the extrinsic cross-link. Thus, it is concluded that the extrinsic cross-link between Lys7 and Lys41, formed by the dinitrophenylene group, does not affect the native conformation of ribonuclease A but destabilizes the denatured conformation, probably by decreasing its chain entropy.