A major question in protein structural analysis concerns the applicability of results from model systems to other proteins. Theoretical approaches seem the best manner of transferring information from one system to another, but their accuracy in the model systems must first be tested with results from experiment. Since bovine pancreatic trypsin inhibitor (BPTI) is a model system for the evaluation of energy minimization and molecular dynamics routines, we can use folding and stability measurements to examine the reliability of these methods. All two-disulfide mutants of BPTI investigated thus far have two very slow folding reactions which have characteristics of proline isomerization. These reactions may occur because the non-native cis form of two of the four prolines in BPTI significantly destabilizes the folded state of the protein. Previous energy minimization studies of wild-type BPTI suggested that the cis form of Pro8 was the most destabilizing of the four prolines [Levitt,M. (1981) J. Mol. Biol., 145, 251–263]. In this paper, we show that mutation of Pro8 - Gln in the two-disulfide bond mutant Val30–Ala51 results in a loss of the slowest folding reaction, consistent with Levitt's prediction.