The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the beta-sheet, and the solvent-exposed face of the alpha-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (Delta DeltaGU-F). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link Delta DeltaGU-F to various structural parameters. The strongest correlation found was between Delta DeltaGU-F and N, the number of methyl(ene) groups within a 6 A radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n = 16,where n is the number of points) was obtained. This increased to 0.81 (n = 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the alpha-helix and beta-sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of alpha-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the alpha-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design.