The X-ray structures of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys119, induced larger changes than did Cys24-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as alpha-helices and beta-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.