Two conformational isomers were observed in the 1H nuclear magnetic resonance (NMR) spectra of the basic pancreatic trypsin inhibitor (BPTI) and of a mutant protein with Gly 36 replaced by Ser, BPTI(G36S). The less abundant isomer differs from the major conformation by different chirality of the Cys 14-Cys 38 disulfide bond. In BPTI, the population of the minor conformer increases from about 1.5% at 4 degrees C to 8% at 68 degrees C. In BPTI(G36S), the population of the minor conformation is about 15% of the total protein, so that a detailed structural study was technically feasible; a trend toward increasing population of the minor conformer at higher temperatures was observed also for this mutant protein. The activation parameters for the exchange between the two conformations were measured in the temperature range 4-68 degrees C, using uniformly 15N-enriched protein samples. Below room temperature the exchange rate of the disulfide flip follows an Arrhenius-type temperature dependence, with negative activation entropy in both proteins. At higher temperatures the exchange rates are governed by a different set of activation parameters, which are similar to those for the ring flips of Tyr 35 about the C beta-C gamma bond. Although the equilibrium enthalpy and entropy were found to be largely temperature independent, the activation entropy changes sign and is positive at higher temperatures. These results suggest that, above room temperature, the disulfide flips are coupled to the same protein structure fluctuations as the ring flips of Tyr 35.