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In order to examine how a protein can be effectively stabilized, two intramolecular cross-links, Glu35-Trp108 and Lys1-His15, which have few unfavorable interactions in the folded state, were simultaneously introduced into hen lysozyme. Both of the intramolecularly cross-linked lysozymes, 35-108 CL and 1-15 CL, containing cross-links Glu35-Trp108 and Lys1-His15, respectively, showed increases in thermal stability of 13.9 and 5.2 degrees C, respectively, over that of wild type, at pH 2.7. On the other hand, a doubly cross-linked lysozyme showed an increase in thermal stability of 20.8 degrees C over that of wild type, under identical conditions. Since the sum of the differences in denaturation temperature between wild type and each of the cross-linked lysozymes was nearly equal to that between wild type and the doubly cross-linked lysozyme, we suggest that the efficient stabilization of the lysozyme molecule was the direct result of the double intramolecular cross-links.